Recombinant IL-5 antagonists useful in treatment of IL-5 mediated disorders

ABSTRACT

Chimeric, humanized and other IL-5 mAbs, derived from high affinity neutralizing mAbs, pharmaceutical compositions containing same, methods of treatment and diagnostics are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. Nos.08/470,110 and 08/467,420, both filed Jun. 6, 1995 which arecontinuation-in-parts of U.S. Ser. No. 08/363,131 filed Dec. 23, 1994.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field ofantibodies and altered antibodies, useful in the treatment and diagnosisof conditions mediated by IL-5 and excess eosinophil production, andmore specifically to mAbs, Fabs, chimeric and humanized antibodies.

BACKGROUND OF THE INVENTION

[0003] Eosinophils have been implicated in the pathogenesis of a widevariety of inflammatory disease states including allergic disordersassociated with hypersensitivity reactions in the lung tissue(Butterfield et al., In: Immunopharmacology of Eosinophils, H. Smith andR. Cook, Eds., p.151-192, Academic Press, London (1993)). A notableexample is asthma, a disease characterized by reversible obstruction ofthe airways leading to non-specific bronchial hyperresponsiveness. Thisin turn is dependent upon the generation of a chronic inflammatoryreaction at the level of the bronchial mucosa and a characteristicinfiltration by macrophages, lymphocytes and eosinophils. The eosinophilappears to play a central role in initiating the mucosal damage typicalof the disease (Corrigan et al., Immunol. Today, 13:501-507 (1992)).Increased numbers of activated eosinophils have been reported in thecirculation, bronchial secretions and lung parenchyma of patients withchronic asthma, and the severity of the disease, as measured by avariety of lung function tests, correlates with blood eosinophil numbers(Griffen et al., J. Aller. Clin. Immunol., 67:548-557 (1991)). Increasednumbers of eosinophils, often in the process of degranulation, have alsobeen recovered in bronchoalveolar lavage (BAL) fluids of patientsundergoing late asthmatic reactions, and reducing eosinophil numbers,usually as a consequence of steroid therapy, is associated withimprovements in clinical symptoms (Bousquet et al., N. Eng. J. Med.,323:1033-1039 (1990)).

[0004] Interleukin 5 (IL-5) is a homodimeric glycoprotein producedpredominantly by activated CD4+ T lymphocytes. In man, IL-5 is largelyresponsible for controlling the growth and differentiation ofeosinophils. Elevated levels of IL-5 are detected in the bronchoalveolarlavage washings of asthmatics (Motojima et al., Allergy, 48:98 (1993)).Mice which are transgenic for IL-5 show a marked eosinophilia inperipheral blood and tissues in the absence of antigenic stimulation(Dent et al., J. Exp. Med., 172:1425 (1990)) and anti-murine IL-5monoclonal antibodies have been shown to have an effect in reducingeosinophilia in the blood and tissues of mice (Hitoshi et al., Int.Immunol., 3:135 (1991)) as well as the eosinophilia associated withparasite infection and allergen challenge in experimental animals(Coffman et al., Science, 245:308-310 (1989), Sher et al., Proc. Natl.Acad. Sci., 83:61-65 (1990), Chand et al., Eur. J. Pharmacol.,211:121-123 (1992)).

[0005] Although corticosteroids are extremely effective in suppressingeosinophil numbers and other inflammatory components of asthma, thereare concerns about their side effects in both severe asthmatics and morerecently in mild to moderate asthmatics. The only other majoranti-inflammatory drug therapies—cromoglycates (cromolyn sodium andnedocromil)—are considerably less effective than corticosteroids andtheir precise mechanism of action remains unknown.

[0006] More recent developments have focused on new inhaled steroids,longer acting bronchodilators and agents acting on novel biochemical orpharmacological targets (e.g., potassium channel activators, leukotrieneantagonists, 5-lipoxygenase (5-LO) inhibitors etc.). An ideal drug wouldbe one that combines the efficacy of steroids with the safety associatedwith cromolyn sodium, yet has increased selectivity and more rapid onsetof action. Neutralizing IL-5 antibodies may potentially be useful inrelieving eosinophila-related symptoms in man.

[0007] Hence there is a need in the art for a high affinity IL-5antagonist, such as a neutralizing monoclonal antibody to humaninterleukin 5, which would reduce eosinophil differentiation andproliferation (i.e., accumulation of eosinophils) and thus eosinophilinflammation.

SUMMARY OF THE INVENTION

[0008] In a first aspect, the present invention provides rodent (e.g.,rat and murine) neutralizing monoclonal antibodies specific for humaninterleukin-5 and having a binding affinity characterized by adissociation constant equal to or less than about 3.5×10⁻¹¹ M asdescribed in the detailed description. Exemplary of such monoclonalantibodies are the murine monoclonal antibodies 2B6, 2E6 and 2F2 and ratmonoclonal antibodies such as 4A6. Another aspect of the invention arehybridomas such as SK119-2B6.206.75(1), SK119-2E3.39.40.2,SK119-2F2.37.80.12, 4A6(1)G1F7 and 5D3(1)F5D6.

[0009] In a related aspect, the present invention provides neutralizingFab fragments or F(ab′)₂ fragments thereof specific for humaninterleukin-5 produced by deleting the Fc region of the rodentneutralizing monoclonal antibodies of the present invention.

[0010] In yet another related aspect, the present invention providesneutralizing Fab fragments or F(ab′)₂ fragments thereof specific forhuman interleukin-5 produced by the chain shuffling technique whereby aheavy (or light) chain immunoglobulin, isolated from rodent neutralizingmonoclonal antibodies of the invention, is expressed with a light chain(or heavy chain, respectively) immunoglobulin library isolated frominterleukin-5 immunized rodents, in a filamentous phage Fab displaylibrary.

[0011] In still another related aspect, the present invention providesan altered antibody specific for human interleukin-5 which comprisescomplementarity determining regions (CDRs) derived from a non-humanneutralizing monoclonal antibody (mAb) characterized by a dissociationconstant equal to or less than about 3.5×10⁻¹¹ M for human interleukin-5and nucleic acid molecules encoding the same. When the altered antibodyis a humanized antibody, the sequences that encode complementaritydetermining regions (CDRs) from a non-human immunoglobulin are insertedinto a first immunoglobulin partner in which at least one, andpreferably all complementarity determining regions (CDRs) of the firstimmunoglobulin partner are replaced by CDRs from the non-humanmonoclonal antibody. Preferably, the first immunoglobulin partner isoperatively linked to a second immunoglobulin partner as well, whichcomprises all or a part of an immuunoglobulin constant chain.

[0012] In a related aspect, the present invention provides CDRs derivedfrom non-human neutralizing monoclonal antibodies (mAbs) characterizedby a dissociation constant equal to or less than about 3.5×10⁻¹¹ M forhuman interleukin-5, and nucleic acid molecules encoding such CDRs.

[0013] In still another aspect, there is provided a chimeric antibodycontaining human heavy and light chain constant regions and heavy andlight chain variable regions derived from non-human neutralizingmonoclonal antibodies characterized by a dissociation constant equal toor less than about 3.5×10⁻¹¹ M for human interleukin-5.

[0014] In yet another aspect, the present invention provides apharmaceutical composition which contains one (or more) of theabove-described altered antibodies and a pharmaceutically acceptablecarrier.

[0015] In a further aspect, the present invention provides a method fortreating conditions in humans associated with excess eosinophilproduction by administering to said human an effective amount of thepharmaceutical composition of the invention.

[0016] In yet another aspect, the present invention provides methodsfor, and components useful in, the recombinant production of alteredantibodies (e.g., engineered antibodies, CDRs, Fab or F(ab)₂ fragments,or analogs thereof) which are derived from non-human neutralizingmonoclonal antibodies (mAbs) characterized by a dissociation constantequal to or less than 3.5×10⁻¹¹ M for human IL-5. These componentsinclude isolated nucleic acid sequences encoding same, recombinantplasmids containing the nucleic acid sequences under the control ofselected regulatory sequences which are capable of directing theexpression thereof in host cells (preferably mammalian) transfected withthe recombinant plasmids. The production method involves culturing atransfected host cell line of the present invention under conditionssuch that an altered antibody, preferably a humanized antibody, isexpressed in said cells and isolating the expressed product therefrom.

[0017] In yet another aspect of the invention is a method to diagnoseconditions associated with excess eosinophil production in a human whichcomprises obtaining a sample of biological fluid from a patient andallowing the antibodies and altered antibodies of the instant inventionto come in contact with such sample under conditions such that anIL-5/(monoclonal or altered) antibody complex is formed and detectingthe presence or absence of said IL-5/antibody complex.

[0018] Other aspects and advantages of the present invention aredescribed further in the detailed description and the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 [SEQ ID NOs: 1 and 15) illustrates the heavy chain variableregion for the murine antibody 2B6, and the murine/human 2B6 chimericantibody. The boxed areas indicate the CDRs.

[0020]FIG. 2 [SEQ ID NOs: 2 and 16] illustrates the light chain variablelegion for the murine antibody 2B6, and the murine/human 2B6 chimericantibody. The boxed areas indicate the CDRs.

[0021]FIG. 3 [SEQ ID NO:3] illustrates the heavy chain variable regionfor the murine antibody 2F2. The boxed areas indicate the CDRs.

[0022]FIG. 4 [SEQ ID NO:4] illustrates the light chain variable regionfor the murine antibody 2F2. The boxed areas indicate the CDRs.

[0023]FIG. 5 [SEQ ID NO:5] illustrates the heavy chain variable regionfor the murine antibody 2E3. The boxed areas indicate the CDRs.

[0024]FIG. 6 [SEQ ID NO:6] illustrates the light chain variable regionfor the murine antibody 2E3. The boxed areas indicate the CDRs.

[0025]FIG. 7 [SEQ ID NOs:7-14] illustrates the heavy and light chainCDRs from murine antibodies 2B6, 2F2 and 2E3.

[0026]FIG. 8 [SEQ ID NOs: 18, 19] illustrates the heavy chain variableregion for the humanized antibody 2B6. The boxed areas indicate theCDRs.

[0027]FIG. 9 [SEQ D NOs: 20, 21] illustrates the light chain variableregion for the humanized antibody 2B6. The boxed areas indicate theCDRs.

[0028]FIG. 10 is a schematic drawing of plasmid pCDIL5HZHC1.0 employedto express a humanized heavy chain gene in mammalian cells. The plasmidcontains a beta lactamase gene (BETA LAC), an SV-40 origin ofreplication (SV40), a cytomegalovirus promoter sequence (CMV), a signalsequence, the humanized heavy chain, a poly A signal from bovine growthhormone (BGH), a betaglobin promoter (beta glopro), a dihydrofolatereductase gene (DHFR), and another BGH sequence poly A signal in a pUC19background.

[0029]FIG. 11 is a schematic drawing of plasmid pCNIL5HZLC1.0 employedto express a humanized light chain gene in mammalian cells.

[0030]FIG. 12 [SEQ ID NOS: 61, 62] illustrates the NewM heavy chainvariable region for the humanized antibody 2B6. The boxed areas indicatethe CDRs.

[0031]FIG. 13 [SEQ ID NOS: 69, 70] illustrates the REI light chainvariable region for the humanized antibody 2B6. The boxed areas indicatethe CDRs.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention provides a variety of antibodies, alteredantibodies and fragments thereof, which are characterized by human IL-5binding specificity, neutralizing activity, and high affinity for humanIL-5 as exemplified in murine monoclonal antibody 2B6. The antibodies ofthe present invention were prepared by conventional hybridomatechniques, phage display combinatorial libraries, immunoglobulin chainshuffling, and humanization techniques to generate novel neutralizingantibodies. These products are useful in therapeutic and pharmaceuticalcompositions for treating IL-5-mediated disorders, e.g., asthma. Theseproducts are also useful in the diagnosis of IL-5-mediated conditions bymeasurement (e.g., enzyme linked immunosorbent assay (ELISA)) ofendogenous IL-5 levels in humans or IL-5 released ex vivo from activatedcells.

[0033] I. Definitions.

[0034] “Altered antibody” refers to a protein encoded by an alteredimmunoglobulin coding region, which may be obtained by expression in aselected host cell. Such altered antibodies are engineered antibodies(e.g., chimeric or humanized antibodies) or antibody fragments lackingall or part of an immunoglobulin constant region, e.g., Fv, Fab, orF(ab)₂ and the like.

[0035] “Altered immunoglobulin coding region” refers to a nucleic acidsequence encoding altered antibody of the invention. When the alteredantibody is a CDR-grafted or humanized antibody, the sequences thatencode the complementarity determining regions (CDRs) from a non-humanimmunoglobulin are inserted into a first immunoglobulin partnercomprising human variable framework sequences. Optionally, the firstimmunoglobulin partner is operatively linked to a second immunoglobulinpartner.

[0036] “First immunoglobulin partner” refers to a nucleic acid sequenceencoding a human framework or human immunoglobulin variable region inwhich the native (or naturally-occurring) CDR-encoding regions arereplaced by the CDR-encoding regions of a donor antibody. The humanvariable region can be an immunoglobulin heavy chain, a light chain (orboth chains), an analog or functional fragments thereof. Such CDRregions, located within the variable region of antibodies(immunoglobulins) can be determined by known methods in the art. Forexample Kabat et al. (Sequences of Proteins of Immunological Interest,4th Ed., U.S. Department of Health and Human Services, NationalInstitutes of Health (1987)) disclose rules for locating CDRs. Inaddition, computer programs are known which are useful for identifyingCDR regions/structures.

[0037] “Neutralizing” refers to an antibody that inhibits IL-5 activityby preventing the binding of human IL-5 to its specific receptor or byinhibiting the signaling of IL-5 through its receptor, should bindingoccur. A mAb is neutralizing if it is 90% effective, preferably 95%effective and most preferably 100% effective in inhibiting IL-5 activityas measured in the B13 cell bioassay (IL-5 Neutralization assay, seeExample 2C).

[0038] The term “high affinity” refers to an antibody having a bindingaffinity characterized by a K_(d) equal to or less than 3.5×10⁻¹¹ M forhuman IL-5 as determined by optical biosensor anaylsis (see Example 2D).

[0039] By “binding specificity for human IL-5” is meant a high affinityfor human, not murine, IL-5.

[0040] “Second immunoglobulin partner” refers to another nucleotidesequence encoding a protein or peptide to which the first immunoglobulinpartner is fused in frame or by means of an optional conventional linkersequence (i.e., operatively linked). Preferably it is an immunoglobulingene. The second immunoglobulin partner may include a nucleic acidsequence encoding the entire constant region for the same (i.e.,homologous—the first and second altered antibodies are derived from thesame source) or an additional (i.e., heterologous) antibody of interest.It may be an immunoglobulin heavy chain or light chain (or both chainsas part of a single polypeptide). The second immunoglobulin partner isnot limited to a particular immunoglobulin class or isotype. Inaddition, the second immunoglobulin partner may comprise part of animmunoglobulin constant region, such as found in a Fab, or F(ab)₂ (i.e.,a discrete part of an appropriate human constant region or frameworkregion). Such second immunoglobulin partner may also comprise a sequenceencoding an integral membrane protein exposed on the outer surface of ahost cell, e.g., as part of a phage display library, or a sequenceencoding a protein for analytical or diagnostic detection, e.g.,horseradish peroxidase, β-galactosidase, etc.

[0041] The terms Fv, Fc, Fd, Fab, or F(ab)₂ are used with their standardmeanings (see, e.g., Harlow et al., Antibodies A Laboratory Manual, ColdSpring Harbor Laboratory, (1988)).

[0042] As used herein, an “engineered antibody” describes a type ofaltered antibody, i.e., a full-length synthetic antibody (e.g., achimeric or humanized antibody as opposed to an antibody fragment) inwhich a portion of the light and/or heavy chain variable domains of aselected acceptor antibody are replaced by analogous parts from one ormore donor antibodies which have specificity for the selected epitope.For example, such molecules may include antibodies characterized by ahumanized heavy chain associated with an unmodified light chain (orchimeric light chain), or vice versa. Engineered antibodies may also becharacterized by alteration of the nucleic acid sequences encoding theacceptor antibody light and/or heavy variable domain framework regionsin order to retain donor antibody binding specificity. These antibodiescan comprise replacement of one or more CDRs (preferably all) from theacceptor antibody with CDRs from a donor antibody described herein.

[0043] A “chimeric antibody” refers to a type of engineered antibodywhich contains naturally-occurring variable region (light chain andheavy chains) derived from a donor antibody in association with lightand heavy chain constant regions derived from an acceptor antibody.

[0044] A “humanized antibody” refers to a type of engineered antibodyhaving its CDRs derived from a non-human donor immunoglobulin, theremaining immunoglobulin-derived parts of the molecule being derivedfrom one (or more) human immunoglobulin(s). In addition, frameworksupport residues may be altered to preserve binding affinity (see, e.g.,Queen et al., Proc. Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson etal., Bio/Technology. 9:421 (1991)).

[0045] The term “donor antibody” refers to an antibody (monoclonal, orrecombinant) which contributes the nucleic acid sequences of itsvariable regions, CDRs, or other functional fragments or analogs thereofto a first immunoglobulin partner, so as to provide the alteredimmunoglobulin coding region and resulting expressed altered antibodywith the antigenic specificity and neutralizing activity characteristicof the donor antibody. One donor antibody suitable for use in thisinvention is a non-human neutralizing monoclonal antibody (i.e., murine)designated as 2B6. The antibody 2B6 is defined as a high affinity,human-IL-5 specific (i.e., does not recognize murine IL-5), neutralizingantibody of isotype IgG, having the variable light chain DNA and aminoacid sequences of SEQ ID NOs: 2 and 16, respectively, and the variableheavy chain DNA and amino acid sequences of SEQ ID NOs: 1 and 15,respectively, on a suitable murine IgG constant region.

[0046] The term “acceptor antibody” refers to an antibody (monoclonal,or recombinant) heterologous to the donor antibody, which contributesall (or any portion, but preferably all) of the nucleic acid sequencesencoding its heavy and/or light chain framework regions and/or its heavyand/or light chain constant regions to the first immunoglobulin partner.Preferably a human antibody is the acceptor antibody.

[0047] “CDRs” are defined as the complementarity determining regionamino acid sequences of an antibody which are the hypervariable regionsof immunoglobulin heavy and light chains. See, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, 4th Ed., U.S.Department of Health and Human Services, National Institutes of Health(1987). There are three heavy chain and three light chain CDRs (or CDRregions) in the variable portion of an immunoglobulin. Thus, “CDRs” asused herein refers to all three heavy chain CDRs, or all three lightchain CDRs (or both all heavy and all light chain CDRs, if appropriate).

[0048] CDRs provide the majority of contact residues for the binding ofthe antibody to the antigen or epitope. CDRs of interest in thisinvention are derived from donor antibody variable heavy and light chainsequences, and include analogs of the naturally occurring CDRs, whichanalogs also share or retain the same antigen binding specificity and/orneutralizing ability as the donor antibody from which they were derived.

[0049] By ‘sharing the antigen binding specificity or neutralizingability’ is meant, for example, that although mAb 2B6 may becharacterized by a certain level of antigen affinity, a CDR encoded by anucleic acid sequence of 2B6 in an appropriate structural environmentmay have a lower, or higher affinity. It is expected that CDRs of 2B6 insuch environments will nevertheless recognize the same epitope(s) as2B6. Exemplary heavy chain CDRs of 2B6 include SEQ ID NO: 7; SEQ ID NO:8; SEQ ID NO: 9; and exemplary light chain CDRs of 2B6 include SEQ IDNO: 10; SEQ ID NO: 11; and SEQ ID NO: 12.

[0050] A “functional fragment” is a partial heavy or light chainvariable sequence (e.g., minor deletions at the amino or carboxyterminus of the immunoglobulin variable region) which retains the sameantigen binding specificity and/or neutralizing ability as the antibodyfrom which the fragment was derived.

[0051] An “analog” is an amino acid sequence modified by at least oneamino acid, wherein said modification can be chemical or a substitutionor a rearrangement of a few amino acids (i.e., no more than 10), whichmodification permits the amino acid sequence to retain the biologicalcharacteristics, e.g., antigen specificity and high affinity, of theunmodified sequence. For example, (silent) mutations can be constructed,via substitutions, when certain endonuclease restriction sites arecreated within or surrounding CDR-encoding regions.

[0052] Analogs may also arise as allelic variations. An “allelicvariation or modification” is an alteration in the nucleic acid sequenceencoding the amino acid or peptide sequences of the invention. Suchvariations or modifications may be due to degeneracy in the genetic codeor may be deliberately engineered to provide desired characteristics.These variations or modifications may or may not result in alterationsin any encoded amino acid sequence.

[0053] The term “effector agents” refers to non-protein carriermolecules to which the altered antibodies, and/or natural or syntheticlight or heavy chains of the donor antibody or other fragments of thedonor antibody may be associated by conventional means. Such non-proteincarriers can include conventional carriers used in the diagnostic field,e.g., polystyrene or other plastic beads, polysaccharides, e.g., as usedin the BIAcore [Pharmacia] system, or other non-protein substancesuseful in the medical field and safe for administration to humans andanimals. Other effector agents may include a macrocycle, for chelating aheavy metal atom, or radioisotopes. Such effector agents may also beuseful to increase the half-life of the altered antibodies, e.g.,polyethylene glycol.

[0054] II. High Affinity IL-5 Monoclonal Antibodies

[0055] For use in constructing the antibodies, altered antibodies andfragments of this invention, a non-human species (for example, bovine,ovine, monkey, chicken, rodent (e.g., murine and rat), etc.) may beemployed to generate a desirable immunoglobulin upon presentment withnative human IL-5 or a peptide epitope therefrom. Conventional hybridomatechniques are employed to provide a hybridoma cell line secreting anon-human mAb to IL-5. Such hybridomas are then screened for bindingusing IL-5 coated to 96-well plates, as described in the Examplessection, or alternatively with biotinylated IL-5 bound to a streptavidincoated plate.

[0056] One exemplary, high affinity, neutralizing mAb of this instantinvention is mAb 2B6, a murine antibody which can be used for thedevelopment of a chimeric or humanized antibody, described in moredetail in Example 1 below. The 2B6 mAb is characterized by an antigenbinding specificity for human IL-5, with a K_(d) of less than 3.5×10⁻¹¹M (about 2.2×10⁻¹¹ M) for IL-5. The K_(d) for IL-5 of a Fab fragmentfrom 2B6 (see, Example 3H) is estimated to be about 9×10⁻¹¹ M asdetermined by optical biosensor. MAb 2B6 appears to block the bindinginteraction between human IL-5 and the α-chain of the human IL-5receptor.

[0057] Another desirable donor antibody is the murine mAb, 2E3. This mAbis characterized by being isotype IgG_(2a), and having a dissociationconstant for hIL-5 of less than 3.5×10⁻¹¹ M (about 2.0×10⁻¹¹ M).

[0058] Yet, another desirable donor antibody is the rat mAb, 4A6. ThismAb is characterized by having a dissociation constant for hIL-5 of lessthan 3.5×10⁻¹¹ M (about 1.8×10⁻¹¹ M). In addition, mAb 4A6 appears toblock the binding interaction between human IL-5 and the β-chain of theIL-5 receptor.

[0059] This invention is not limited to the use of the 2B6 mAb, the 2E3mAb, or its hypervariable (i.e., CDR) sequences. Any other appropriatehigh affinity IL-5 antibodies characterized by a dissociation constantequal or less than 3.5×10⁻¹¹ M for human IL-5 and correspondinganti-IL-5 CDRs may be substituted therefor. Wherever in the followingdescription the donor antibody is identified as 2B6 or 2E3, thisdesignation is made for illustration and simplicity of description only.

[0060] III. Antibody Fragments

[0061] The present invention also includes the use of Fab fragments orF(ab′)₂ fragments derived from mAbs directed against human IL-5. Thesefragments are useful as agents protective in vivo against IL-5 andeosinophil-mediated conditions or in vitro as part of an IL-5diagnostic. A Fab fragment contains the entire light chain and aminoterminal portion of the heavy chain; and an F(ab′)₂ fragment is thefragment formed by two Fab fragments bound by disulfide bonds. MAbs 2B6,2E3, and other similar high affinity, IL-5 binding antibodies, providesources of Fab fragments and F(ab′)₂ fragments which can be obtained byconventional means, e.g., cleavage of the mAb with the appropriateproteolytic enzymes, papain and/or pepsin, or by recombinant methods.These Fab and F(ab′)₂ fragments are useful themselves as therapeutic,prophylactic or diagnostic agents, and as donors of sequences includingthe variable regions and CDR sequences useful in the formation ofrecombinant or humanized antibodies as described herein.

[0062] The Fab and F(ab′)₂ fragments can be constructed via acombinatorial phage library (see, e.g., Winter et al., Ann. Rev.Immunol., 12:433455 (1994)) or via immunoglobulin chain shuffling (see,e.g., Marks et al., Bio/Technology, 10:779-783 (1992), which are bothhereby incorporated by reference in their entirety) wherein the Fd orV_(H) immunoglobulin from a selected antibody (e.g., 2B6) is allowed toassociate with a repertoire of light chain immunoglobulins, V_(L) (orV_(K)), to form novel Fabs. Conversely, the light chain immunoglobulinfrom a selected antibody may be allowed to associate with a repertoireof heavy chain immunoglobulins, V_(H) (or Fd), to form novel Fabs.Neutralizing IL-5 Fabs were obtained when the Fd of mAb 2B6 was allowedto associate with a repertoire of light chain immunoglobulins, asdescribed in more detail in the Examples section. Hence, one is able torecover neutralizing Fabs with unique sequences (nucleotide and aminoacid) from the chain shuffling technique.

[0063] IV. Anti-IL-5 Amino Acid and Nucleotide Sequences of Interest

[0064] The mAb 2B6 or other antibodies described above may contributesequences, such as variable heavy and/or light chain peptide sequences,framework sequences, CDR sequences, functional fragments, and analogsthereof, and the nucleic acid sequences encoding them, useful indesigning and obtaining various altered antibodies which arecharacterized by the antigen binding specificity of the donor antibody.

[0065] As one example, the present invention thus provides variablelight chain and variable heavy chain sequences from the IL-5 murineantibody 2B6 and sequences derived therefrom. The heavy chain variableregion of 2B6 is illustrated by FIG. 1. The CDR-encoding regions areindicated by the boxed areas and are provided in SEQ ID NO: 7; SEQ IDNO: 8; and SEQ ID NO: 9. The light chain clone variable region of 2B6 isillustrated by FIG. 2. The CDR-encoding regions are provided in SEQ IDNO: 10; SEQ ID NO: 11; and SEQ ID NO: 12.

[0066] A humanized heavy chain variable region is illustrated in FIG. 8(SEQ ID NOs: 18 and 19]. The signal sequence is also provided in SEQ IDNO: 17. Other suitable signal sequences, known to those of skill in theart, may be substituted for the signal sequences exemplified herein. TheCDR amino acid sequences of this construct are identical to the nativemurine and chimeric heavy chain CDRs and are provided by SEQ ID NO: 7,SEQ ID NO: 8, and SEQ ID NO: 9. An exemplary (synthetic) humanized lightchain variable sequence is illustrated in FIG. 9 [SEQ ID NOs: 20 and21].

[0067] The nucleic acid sequences of this invention, or fragmentsthereof, encoding the variable light chain and heavy chain peptidesequences are also useful for mutagenic introduction of specific changeswithin the nucleic acid sequences encoding the CDRs or frameworkregions, and for incorporation of the resulting modified or fusionnucleic acid sequence into a plasmid for expression. For example, silentsubstitutions in the nucleotide sequence of the framework andCDR-encoding regions were used to create restriction enzyme sites whichfacilitated insertion of mutagenized CDR (and/or framework) regions.These CDR-encoding regions were used in the construction of a humanizedantibody of this invention.

[0068] Taking into account the degeneracy of the genetic code, variouscoding sequences may be constructed which encode the variable heavy andlight chain amino acid sequences, and CDR sequences of the invention aswell as functional fragments and analogs thereof which share the antigenspecificity of the donor antibody. The isolated nucleic acid sequencesof this invention, or fragments thereof, encoding the variable chainpeptide sequences or CDRs can be used to produce altered antibodies,e.g., chimeric or humanized antibodies, or other engineered antibodiesof this invention when operatively combined with a second immunoglobulinpartner.

[0069] It should be noted that in addition to isolated nucleic acidsequences encoding portions of the altered antibody and antibodiesdescribed herein, other such nucleic acid sequences are encompassed bythe present invention, such as those complementary to the nativeCDR-encoding sequences or complementary to the modified human frameworkregions surrounding the CDR-encoding regions. Useful DNA sequencesinclude those sequences which hybridize under stringent hybridizationconditions [see, T. Maniatis et al, Molecular Cloning (A LaboratoryManual), Cold Spring Harbor Laboratory (1982), pages 387 to 389] to theDNA sequences. An example of one such stringent hybridization conditionis hybridization at 4×SSC at 65° C., followed by a washing in 0.1×SSC at65° C. for an hour. Alternatively an exemplary stringent hybridizationcondition is in 50% formamide, 4×SSC at 42° C. Preferably, thesehybridizing DNA sequences are at least about 18 nucleotides in length,i.e., about the size of a CDR.

[0070] V. Altered Immunoglobulin Molecules and Altered Antibodies

[0071] Altered immunoglobulin molecules can encode altered antibodieswhich include engineered antibodies such as chimeric antibodies andhumanized antibodies. A desired altered immunoglobulin coding regioncontains CDR-encoding regions that encode peptides having the antigenspecificity of an IL-5 antibody, preferably a high affinity antibodysuch as provided by the present invention, inserted into a firstimmunoglobulin partner (a human framework or human immunoglobulinvariable region).

[0072] Preferably, the first immunoglobulin partner is operativelylinked to a second immunoglobulin partner. The second immunoglobulinpartner is defined above, and may include a sequence encoding a secondantibody region of interest, for example an Fc region. Secondimmunoglobulin partners may also include sequences encoding anotherimmunoglobulin to which the light or heavy chain constant region isfused in frame or by means of a linker sequence. Engineered antibodiesdirected against functional fragments or analogs of IL-5 may be designedto elicit enhanced binding with the same antibody.

[0073] The second immunoglobulin partner may also be associated witheffector agents as defined above, including non-protein carriermolecules, to which the second immunoglobulin partner may be operativelylinked by conventional means.

[0074] Fusion or linkage between the second immunoglobulin partners,e.g., antibody sequences, and the effector agent may be by any suitablemeans, e.g., by conventional covalent or ionic bonds, protein fusions,or hetero-bifunctional cross-linkers, e.g., carbodiimide,glutaraldehyde, and the like. Such techniques are known in the art andreadily described in conventional chemistry and biochemistry texts.

[0075] Additionally, conventional linker sequences which simply providefor a desired amount of space between the second immunoglobulin partnerand the effector agent may also be constructed into the alteredimmunoglobulin coding region. The design of such linkers is well knownto those of skill in the art.

[0076] In addition, signal sequences for the molecules of the inventionmay be modified to enhance expression. As one example the 2B6 humanizedantibody having the signal sequence and CDRs derived from the murineheavy chain sequence, had the original signal peptide replaced withanother signal sequence [SEQ ID NO: 17].

[0077] An exemplary altered antibody contains a variable heavy and/orlight chain peptide or protein sequence having the antigen specificityof mAb 2B6, e.g., the V_(H) and V_(L) chains. Still another desirablealtered antibody of this invention is characterized by the amino acidsequence containing at least one, and preferably all of the CDRs of thevariable region of the heavy and/or light chains of the murine antibodymolecule 2B6 with the remaining sequences being derived from a humansource, or a functional fragment or analog thereof. See, e.g., thehumanized V_(H) and V, regions (FIGS. 8 and 9).

[0078] In still a further embodiment, the engineered antibody of theinvention may have attached to it an additional agent. For example, theprocedure of recombinant DNA technology may be used to produce anengineered antibody of the invention in which the Fc fragment or CH2 CH3domain of a complete antibody molecule has been replaced by an enzyme orother detectable molecule (i.e., a polypeptide effector or reportermolecule).

[0079] The second immunoglobulin partner may also be operatively linkedto a non-immunoglobulin peptide, protein or fragment thereofheterologous to the CDR-containing sequence having the antigenspecificity of murine 2B6. The resulting protein may exhibit bothanti-IL-5 antigen specificity and characteristics of thenon-immunoglobulin upon expression. That fusion partner characteristicmay be, e.g., a functional characteristic such as another binding orreceptor domain, or a therapeutic characteristic if the fusion partneris itself a therapeutic protein, or additional antigeniccharacteristics.

[0080] Another desirable protein of this invention may comprise acomplete antibody molecule, having full length heavy and light chains,or any discrete fragment thereof, such as the Fab or F(ab′)₂ fragments,a heavy chain dimer, or any minimal recombinant fragments thereof suchas an F, or a single-chain antibody (SCA) or any other molecule with thesame specificity as the selected donor mAb, e.g., mAb 2B6 or 2E3. Suchprotein may be used in the form of an altered antibody, or may be usedin its unfused form.

[0081] Whenever the second immunoglobulin partner is derived from anantibody different from the donor antibody, e.g., any isotype or classof immunoglobulin framework or constant regions, an engineered antibodyresults. Engineered antibodies can comprise immunoglobulin (Ig) constantregions and variable framework regions from one source, e.g., theacceptor antibody, and one or more (preferably all) CDRs from the donorantibody, e.g., the anti-IL-5 antibody described herein. In addition,alterations, e.g., deletions, substitutions, or additions, of theacceptor mAb light and/or heavy variable domain framework region at thenucleic acid or amino acid levels, or the donor CDR regions may be madein order to retain donor antibody antigen binding specificity.

[0082] Such engineered antibodies are designed to employ one (or both)of the variable heavy and/or light chains of the IL-5 mAb (optionallymodified as described) or one or more of the below-identified heavy orlight chain CDRs (see also FIG. 7). The engineered antibodies of theinvention are neutralizing, i.e., they desirably block binding to thereceptor of the IL-5 protein and they also block or preventproliferation of IL-5 dependent cells.

[0083] Such engineered antibodies may include a humanized antibodycontaining the framework regions of a selected human immunoglobulin orsubtype, or a chimeric antibody containing the human heavy and lightchain constant regions fused to the IL-5 antibody functional fragments.A suitable human (or other animal) acceptor antibody may be one selectedfrom a conventional database, e.g., the KABAT® database, Los Alamosdatabase, and Swiss Protein database, by homology to the nucleotide andamino acid sequences of the donor antibody. A human antibodycharacterized by a homology to the framework regions of the donorantibody (on an amino acid basis) may be suitable to provide a heavychain constant region and/or a heavy chain variable framework region forinsertion of the donor CDRs. A suitable acceptor antibody capable ofdonating light chain constant or variable framework regions may beselected in a similar manner. It should be noted that the acceptorantibody heavy and light chains are not required to originate from thesame acceptor antibody.

[0084] Desirably the heterologous framework and constant regions areselected from human immunoglobulin classes and isotypes, such as IgG(subtypes 1 through 4), IgM, IgA, and IgE. However, the acceptorantibody need not comprise only human immunoglobulin protein sequences.For instance a gene may be constructed in which a DNA sequence encodingpart of a human immunoglobulin chain is fused to a DNA sequence encodinga non-immunoglobulin amino acid sequence such as a polypeptide effectoror reporter molecule.

[0085] One example of a particularly desirable humanized antibodycontains CDRs of 2B6 inserted onto the framework regions of a selectedhuman antibody sequence. For neutralizing humanized antibodies, one, twoor preferably three CDRs from the IL-5 antibody heavy chain and/or lightchain variable regions are inserted into the framework regions of theselected human antibody sequence, replacing the native CDRs of thelatter antibody.

[0086] Preferably, in a humanized antibody, the variable domains in bothhuman heavy and light chains have been engineered by one or more CDRreplacements. It is possible to use all six CDRs, or variouscombinations of less than the six CDRs. Preferably all six CDRs arereplaced. It is possible to replace the CDRs only in the human heavychain, using as light chain the unmodified light chain from the humanacceptor antibody. Still alternatively, a compatible light chain may beselected from another human antibody by recourse to the conventionalantibody databases. The remainder of the engineered antibody may bederived from any suitable acceptor human immunoglobulin.

[0087] The engineered humanized antibody thus preferably has thestructure of a natural human antibody or a fragment thereof, andpossesses the combination of properties required for effectivetherapeutic use, e.g., treatment of IL-5 mediated inflammatory diseasesin man, or for diagnostic uses.

[0088] As another example, an engineered antibody may contain three CDRsof the variable light chain region of 2E3 [SEQ ID NO: 10, 11 and 13] andthree CDRs of the variable heavy chain region of 2B6 [SEQ ID NO: 7, 8and 9]. The resulting humanized antibody should be characterized by thesame antigen binding specificity and high affinity of mAb 2B6.

[0089] It will be understood by those skilled in the art that anengineered antibody may be further modified by changes in variabledomain amino acids without necessarily affecting the specificity andhigh affinity of the donor antibody (i.e., an analog). It is anticipatedthat heavy and light chain amino acids may be substituted by other aminoacids either in the variable domain frameworks or CDRs or both.

[0090] In addition, the constant region may be altered to enhance ordecrease selective properties of the molecules of the instant invention.For example, dimerization, binding to Fc receptors, or the ability tobind and activate complement (see, e.g., Angal et al., Mol. Immunol,30:105-108 (1993), Xu et al., J. Biol. Chem, 269:3469-3474 (1994),Winter et al., EP 307,434-B).

[0091] An altered antibody which is a chimeric antibody differs from thehumanized antibodies described above by providing the entire non-humandonor antibody heavy chain and light chain variable regions, includingframework regions, in association with human immunoglobulin constantregions for both chains. It is anticipated that chimeric antibodieswhich retain additional non-human sequence relative to humanizedantibodies of this invention may elicit a significant immune response inhumans.

[0092] Such antibodies are useful in the prevention and treatment ofIL-5 mediated disorders, as discussed below.

[0093] VI. Production of Altered Antibodies and Engineered Antibodies

[0094] Preferably, the variable light and/or heavy chain sequences andthe CDRs of mAb 2B6 or other suitable donor mAbs (e.g., 2E3, 2F2, 4A6,etc.), and their encoding nucleic acid sequences, are utilized in theconstruction of altered antibodies, preferably humanized antibodies, ofthis invention, by the following process. The same or similar techniquesmay also be employed to generate other embodiments of this invention,

[0095] A hybridoma producing a selected donor mAb, e.g., the murineantibody 2B6, is conventionally cloned, and the DNA of its heavy andlight chain variable regions obtained by techniques known to one ofskill in the art, e.g., the techniques described in Sambrook et al.,(Molecular Cloning (A Laboratory Manual), 2nd edition, Cold SpringHarbor Laboratory (1989)). The variable heavy and light regions of 2B6containing at least the CDR-encoding regions and those portions of theacceptor mAb light and/or heavy variable domain framework regionsrequired in order to retain donor mAb binding specificity, as well asthe remaining immunoglobulin-derived parts of the antibody chain derivedfrom a human immunoglobulin are obtained using polynucleotide primersand reverse transcriptase. The CDR-encoding regions are identified usinga known database and by comparison to other antibodies.

[0096] A mouse/human chimeric antibody may then be prepared and assayedfor binding ability. Such a chimeric antibody contains the entirenon-human donor antibody V_(H) and V_(L) regions, in association withhuman Ig constant regions for both chains.

[0097] Homologous framework regions of a heavy chain variable regionfrom a human antibody were identified using computerized databases,e.g., KABAT®, and a human antibody having homology to 2B6 was selectedas the acceptor antibody. The sequences of synthetic heavy chainvariable regions containing the 2B6 CDR-encoding regions within thehuman antibody frameworks were designed with optional nucleotidereplacements in the framework regions to incorporate restriction sites.This designed sequence was then synthesized using long syntheticoligomers. Alternatively, the designed sequence can be synthesized byoverlapping oligonucleotides, amplified by polymerase chain reaction(PCR), and corrected for errors.

[0098] A suitable light chain variable framework region was designed ina similar manner.

[0099] A humanized antibody may be derived from the chimeric antibody,or preferably, made synthetically by inserting the donor mAbCDR-encoding regions from the heavy and light chains appropriatelywithin the selected heavy and light chain framework. Alternatively, ahumanized antibody of the invention made be prepared using standardmutagenesis techniques. Thus, the resulting humanized antibody containshuman framework regions and donor mAb CDR-encoding regions. There may besubsequent manipulation of framework residues. The resulting humanizedantibody can be expressed in recombinant host cells, e.g., COS, CHO ormyeloma cells. Other humanized antibodies may be prepared using thistechnique on other suitable IL-5-specific, neutralizing, high affinity,non-human antibodies.

[0100] A conventional expression vector or recombinant plasmid isproduced by placing these coding sequences for the altered antibody inoperative association with conventional regulatory control sequencescapable of controlling the replication and expression in, and/orsecretion from, a host cell. Regulatory sequences include promotersequences, e.g., CMV promoter, and signal sequences, which can bederived from other known antibodies. Similarly, a second expressionvector can be produced having a DNA sequence which encodes acomplementary antibody light or heavy chain. Preferably this secondexpression vector is identical to the first except insofar as the codingsequences and selectable markers are concerned, so to ensure as far aspossible that each polypeptide chain is functionally expressed.Alternatively, the heavy and light chain coding sequences for thealtered antibody may reside on a single vector.

[0101] A selected host cell is co-transfected by conventional techniqueswith both the first and second vectors (or simply transfected by asingle vector) to create the transfected host cell of the inventioncomprising both the recombinant or synthetic light and heavy chains. Thetransfected cell is then cultured by conventional techniques to producethe engineered antibody of the invention. The humanized antibody whichincludes the association of both the recombinant heavy chain and/orlight chain is screened from culture by appropriate assay, such as ELISAor RIA. Similar conventional techniques may be employed to constructother altered antibodies and molecules of this invention.

[0102] Suitable vectors for the cloning and subcloning steps employed inthe methods and construction of the compositions of this invention maybe selected by one of skill in the art. For example, the conventionalpUC series of cloning vectors, may be used. One vector used is pUC 19,which is commercially available from supply houses, such as Amersham(Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden).Additionally, any vector which is capable of replicating readily, has anabundance of cloning sites and selectable genes (e.g., antibioticresistance), and is easily manipulated may be used for cloning. Thus,the selection of the cloning vector is not a limiting factor in thisinvention.

[0103] Similarly, the vectors employed for expression of the engineeredantibodies according to this invention may be selected by one of skillin the art from any conventional vector. The vectors also containselected regulatory sequences (such as CMV promoters) which direct thereplication and expression of heterologous DNA sequences in selectedhost cells. These vectors contain the above described DNA sequenceswhich code for the engineered antibody or altered immunoglobulin codingregion. In addition, the vectors may incorporate the selectedimmunoglobulin sequences modified by the insertion of desirablerestriction sites for ready manipulation.

[0104] The expression vectors may also be characterized by genessuitable for amplifying expression of the heterologous DNA sequences,e.g., the mammalian dihydrofolate reductase gene (DHFR). Otherpreferable vector sequences include a poly A signal sequence, such asfrom bovine growth hormone (BGH) and the betaglobin promoter sequence(betaglopro). The expression vectors useful herein may be synthesized bytechniques well known to those skilled in this art.

[0105] The components of such vectors, e.g. replicons, selection genes,enhancers, promoters, signal sequences and the like, may be obtainedfrom commercial or natural sources or synthesized by known proceduresfor use in directing the expression and/or secretion of the product ofthe recombinant DNA in a selected host. Other appropriate expressionvectors of which numerous types are known in the art for mammalian,bacterial, insect, yeast, and fungal expression may also be selected forthis purpose.

[0106] The present invention also encompasses a cell line transfectedwith a recombinant plasmid containing the coding sequences of theengineered antibodies or altered immunoglobulin molecules thereof. Hostcells useful for the cloning and other manipulations of these cloningvectors are also conventional. However, most desirably, cells fromvarious strains of E. coli are used for replication of the cloningvectors and other steps in the construction of altered antibodies ofthis invention.

[0107] Suitable host cells or cell lines for the expression of theengineered antibody or altered antibody of the invention are preferablymammalian cells such as CHO, COS, a fibroblast cell (e.g., 3T3), andmyeloid cells, and more preferably a CHO or a myeloid cell. Human cellsmay be used, thus enabling the molecule to be modified with humanglycosylation patterns. Alternatively, other eukaryotic cell lines maybe employed. The selection of suitable mammalian host cells and methodsfor transformation, culture, amplification, screening and productproduction and purification are known in the art. See, e.g., Sambrook etal., cited above.

[0108] Bacterial cells may prove useful as host cells suitable for theexpression of the recombinant Fabs of the present invention (see, e.g.,Plückthun, A., Immunol. Rev., 130:151-188 (1992)). However, due to thetendency of proteins expressed in bacterial cells to be in an unfoldedor improperly folded form or in a non-glycosylated form, any recombinantFab produced in a bacterial cell would have to be screened for retentionof antigen binding ability. If the molecule expressed by the bacterialcell was produced in a properly folded form, that bacterial cell wouldbe a desirable host. For example, various strains of E. coli used forexpression are well-known as host cells in the field of biotechnology.Various strains of B. subtilis, Streptomyces, other bacilli and the likemay also be employed in this method.

[0109] Where desired, strains of yeast cells known to those skilled inthe art are also available as host cells, as well as insect cells, e.g.Drosophila and Lepidoptera and viral expression systems. See, e.g.Miller et al., Genetic Engineering, 8:277-298, Plenum Press (1986) andreferences cited therein.

[0110] The general methods by which the vectors of the invention may beconstructed, the transfection methods required to produce the host cellsof the invention, and culture methods necessary to produce the alteredantibody of the invention from such host cell are all conventionaltechniques. Likewise, once produced, the altered antibodies of theinvention may be purified from the cell culture contents according tostandard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like. Such techniques are within the skill ofthe art and do not limit this invention.

[0111] Yet another method of expression of the humanized antibodies mayutilize expression in a transgenic animal, such as described in U.S.Pat. No. 4,873,316. This relates to an expression system using theanimal's casein promoter which when transgenically incorporated into amammal permits the female to produce the desired recombinant protein inits milk.

[0112] Once expressed by the desired method, the engineered antibody isthen examined for in vitro activity by use of an appropriate assay.Presently conventional ELISA assay formats are employed to assessqualitative and quantitative binding of the engineered antibody to IL-5.Additionally, other in vitro assays may also be used to verifyneutralizing efficacy prior to subsequent human clinical studiesperformed to evaluate the persistence of the engineered antibody in thebody despite the usual clearance mechanisms.

[0113] Following the procedures described for humanized antibodiesprepared from 2B6, one of skill in the art may also construct humanizedantibodies from other donor IL-5 antibodies, variable region sequencesand CDR peptides described herein. Engineered antibodies can be producedwith variable region frameworks potentially recognized as “self” byrecipients of the engineered antibody. Minor modifications to thevariable region frameworks can be implemented to effect large increasesin antigen binding without appreciable increased immunogenicity for therecipient. Such engineered antibodies may effectively treat a human forIL-5 mediated conditions. Such antibodies may also be useful in thediagnosis of such conditions.

[0114] VII. Therapeuric/Prophylactic Uses

[0115] This invention also relates to a method of treating humansexperiencing eosinophilia-related symptoms, such as asthma, whichcomprises administering an effective dose of antibodies including one ormore of the engineered antibodies or altered antibodies describedherein, or fragments thereof.

[0116] The therapeutic response induced by the use of the molecules ofthis invention is produced by the binding to human IL-5 and thussubsequently blocking eosinophil stimulation. Thus, the molecules of thepresent invention, when in preparations and formulations appropriate fortherapeutic use, are highly desirable for those persons experiencing anallergic and/or atopic response, or a response associated witheosinophilia, such as but not limited to, allergic rhinitis, asthma,chronic eosinophilic pneumonia, allergic bronchopulmonary aspergillosis,coeliac disease, eosinophilic gastroenteritis, Churg-Strauss syndrome(periarteritis nodosa plus atopy), eosinophilic myalgia syndrome,hypereosinophilic syndrome, oedematous reactions including episodicangiodema, helminth infections, where eosinophils may have a protectiverole, onchocercal dermatitis and atopic dermatitis.

[0117] The altered antibodies, antibodies and fragments thereof of thisinvention may also be used in conjunction with other antibodies,particularly human mAbs reactive with other markers (epitopes)responsible for the condition against which the engineered antibody ofthe invention is directed.

[0118] The therapeutic agents of this invention are believed to bedesirable for treatment of allergic conditions from about 2 days toabout 3 weeks, or as needed. For example, longer treatments may bedesirable when treating seasonal rhinitis or the like. This represents aconsiderable advance over the currently used infusion protocol withprior art treatments of IL-5 mediated disorders. The dose and durationof treatment relates to the relative duration of the molecules of thepresent invention in the human circulation, and can be adjusted by oneof skill in the art depending upon the condition being treated and thegeneral health of the patient.

[0119] The mode of administration of the therapeutic agent of theinvention may be any suitable route which delivers the agent to thehost. The altered antibodies, antibodies, engineered antibodies, andfragments thereof, and pharmaceutical compositions of the invention areparticularly useful for parenteral administration, i.e., subcutaneously,intramuscularly, intravenously, or intranasally.

[0120] Therapeutic agents of the invention may be prepared aspharmaceutical compositions containing an effective amount of theengineered (e.g., humanized) antibody of the invention as an activeingredient in a pharmaceutically acceptable carrier. In the prophylacticagent of the invention, an aqueous suspension or solution containing theengineered antibody, preferably buffered at physiological pH, in a formready for injection is preferred. The compositions for parenteraladministration will commonly comprise a solution of the engineeredantibody of the invention or a cocktail thereof dissolved in anpharmaceutically acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be employed, e.g., 0.4% saline, 0.3%glycine, and the like. These solutions are sterile and generally free ofparticulate matter. These solutions may be sterilized by conventional,well known sterilization techniques (e.g., filtration). The compositionsmay contain pharmaceutically acceptable auxiliary substances as requiredto approximate physiological conditions such as pH adjusting andbuffering agents, etc. The concentration of the antibody of theinvention in such pharmaceutical formulation can vary widely, i.e., fromless than about 0.5%, usually at or at least about 1% to as much as 15or 20% by weight and will be selected primarily based on fluid volumes,viscosities, etc., according to the particular mode of administrationselected.

[0121] Thus, a pharmaceutical composition of the invention forintramuscular injection could be prepared to contain 1 mL sterilebuffered water, and between about 1 ng to about 100 mg, e.g. about 50 ngto about 30 mg or more preferably, about 5 mg to about 25 mg, of anengineered antibody of the invention. Similarly, a pharmaceuticalcomposition of the invention for intravenous infusion could be made upto contain about 250 ml of sterile Ringer's solution, and about 1 toabout 30 and preferably 5 mg to about 25 mg of an engineered antibody ofthe invention. Actual methods for preparing parenterally administrablecompositions are well known or will be apparent to those skilled in theart and are described in more detail in, for example, Remington'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.

[0122] It is preferred that the therapeutic agent of the invention, whenin a pharmaceutical preparation, be present in unit dose forms. Theappropriate therapeutically effective dose can be determined readily bythose of skill in the art. To effectively treat an inflammatory disorderin a human or other animal, one dose of approximately 0.1 mg toapproximately 20 mg per 70 kg body weight of a protein or an antibody ofthis invention should be administered parenterally, preferably i.v. ori.m. (intramuscularly). Such dose may, if necessary, be repeated atappropriate time intervals selected as appropriate by a physician duringthe inflammatory response.

[0123] The altered antibodies and engineered antibodies of thisinvention may also be used in diagnostic regimens, such as for thedetermination of IL-5 mediated disorders or tracking progress oftreatment of such disorders. As diagnostic reagents, these alteredantibodies may be conventionally labeled for use in ELISA's and otherconventional assay formats for the measurement of IL-5 levels in serum,plasma or other appropriate tissue, or the release by human cells inculture. The nature of the assay in which the altered antibodies areused are conventional and do not limit this disclosure.

[0124] Thus, one embodiment of the present invention relates to a methodfor aiding the diagnosis of allergies and other conditions associatedwith excess eosinophil production in a patient which comprises the stepsof determining the amount of human IL-5 in sample (plasma or tissue)obtained from said patient and comparing said determined amount to themean amount of human IL-5 in the normal population, whereby the presenceof a significantly elevated amount of IL-5 in the patient's sample is anindication of allergies and other conditions associated with excesseosinophil production.

[0125] The antibodies, altered antibodies or fragments thereof describedherein can be lyophilized for storage and reconstituted in a suitablecarrier prior to use. This technique has been shown to be effective withconventional immunoglobulins and art-known lyophilization andreconstitution techniques can be employed.

[0126] The following examples illustrate various aspects of thisinvention including the construction of exemplary engineered antibodiesand expression thereof in suitable vectors and host cells, and are notto be construed as limiting the scope of this invention. All amino acidsare identified by conventional three letter or single letter codes. Allnecessary restriction enzymes, plasmids, and other reagents andmaterials were obtained from commercial sources unless otherwiseindicated. All general cloning ligation and other recombinant DNAmethodology were as performed in T. Maniatis et al., cited above, or thesecond edition thereof (1989), eds. Sambrook et al., by the samepublisher (“Sambrook et al.”).

EXAMPLE 1 Production of MAbs to hIL-5

[0127] Human IL-5 was expressed in Drosophila Schneider 2 (S₂) cells andpurified to homogeneity. Murine IL-5 was expressed in Baculovirus usingSpodoptera frugiperda 21 (Sf21) cells and purified to homogeneity.Monoclonal antibody TRFK-5 (a neutralizing rat anti-mouse IL-5 antibody)was obtained from Genzyme Corp. (Cambridge, Mass.).

[0128] A. Immunization Procedure:

[0129] Recombinant human IL-5 (IL-5) was used as the immunogen for apanel of seven CAF₁ female mice (Charles River, Wilmington, Mass.). Theanimals received three subcutaneous injections of IL-5 in phosphatebuffered saline (PBS) emulsified with a one to one ratio of TiterMAX™(CytoRx Corp., Norcross, Ga.) over a period of four months. The primingantigen dose was 50 μg (micrograms) and boosts were 25 and 10 μg(micrograms). After the boosts, serum samples were collected and assayedboth for binding to IL-5 and for neutralization activity via thereceptor binding inhibition assay and B13 proliferation assay (or IL-5neutralization assay (Example 2C)). All of the mice produced serumsamples that bound to IL-5. Animals selected as spleen donors wereboosted intravenously with 10 μg (micrograms) of recombinant human IL-5three days prior to euthanasia.

[0130] B. Hybridoma Development:

[0131] The fusion procedure, first reported by Kohler et al., (Nature,256:495 (1975)), was used with modifications to perform the techniqueusing a cell monolayer (Kennet et al., Eds., “Hybridomas: A newdimension in biological analysis”, pp. 368-377, Plenum Press, New York).Spleen cells from two donor mice were pooled and fusions performed usinga ratio of 50 million spleen cells to ten million SP2/0/Ag14 myelomacells. Supernatants from fusion-positive wells were assayed for bindingto IL-5 by ELISA. Wells containing cells producing antibody to IL-5 wereexpanded and supernatants screened in an IL-5 receptor bindinginhibition assay, and a B13 (neutralization) proliferation assay(described below).

[0132] Sixteen hybridomas were isolated which secreted mAbs reactivewith IL-5. The hybridoma supernatants were mixed with iodinated IL-5,added to a membrane extract prepared from Drosophila cells expressingthe α-chain of the IL-5 receptor (IL-5R), and assayed for inhibition ofreceptor binding. Eleven of the hybridoma supernatants inhibited bygreater than 60% the binding of iodinated IL-5 to the IL-5 receptorα-chain. Three of the mAbs, 2B6, 2E3 and 2F2, also inhibited by greaterthan 70% the proliferation of murine B13 cells in response to human butnot murine IL-5. Five of the hybridomas, four of which blocked bindingand/or proliferation (1C6, 2B6, 2E3 and 2F2) and 1 of which wasnon-neutralizing (24G9), were repeatedly subcloned in soft agar togenerate stable clonal cell lines. Supernatants from the cloned lineswere screened for cross-reactivity by ELISA and did not bind to humanIL-1α, IL-1β, IL-4, IL-8, M-CSF or TGFα. The mAbs were purified andbinding affinities were estimated from optical biosensor (BIAcore)analysis to range from 10 to 100 pM. Supernatants from the lines wereisotyped using murine isotyping reagents (PharMingen, San Diego,Calif.). A summary of the affinities and IC₅₀ for neutralizingactivities of the mAbs is presented in Table I (Example 2).

[0133] By similar methods, rat hybridomas were derived from immunizedrats, using a comparable immunization protocol and rat myelomas for thefusion as described for the mouse. Two rat hybridomas, 4A6 and 5D3, wereidentified that produced mAbs which bound to IL-5. Like mAbs 2B6, 2E3and 2F2, mAbs 4A6 and 5D3 were found to be neutralizing in the B13 assaydescribed below.

[0134] C. Hybridoma Deposit:

[0135] The hybridoma cell line SK119-2B6.206.75(1) producing monoclonalantibody 2B6 was deposited with the American Type Culture Collection(ATCC), Rockville, Md., USA, under accession number HB 11783, and hasbeen accepted as a patent deposit, in accordance with the BudapestTreaty of 1977 governing the deposit of microorganisms for the purposesof patent procedure.

[0136] The hybridoma cell line SK119-2E3.39.40.2 producing monoclonalantibody 2E3 was deposited with the American Type Culture Collection(ATCC), Rockville, Md., USA, under accession number HB 11782, and hasbeen accepted as a patent deposit, in accordance with the BudapestTreaty of 1977 governing the deposit of microorganisms for the purposesof patent procedure.

[0137] The hybridoma cell line SK 119-2F2.37.80.12 producing monoclonalantibody 2F2 was deposited with the American Type Culture Collection(ATCC), Rockville, Md., USA, under accession number HB 11781, and hasbeen accepted as a patent deposit, in accordance with the BudapestTreaty of 1977 governing the deposit of microorganisms for the purposesof patent procedure.

[0138] The hybridoma cell line SK119-24G9.8.20.5 producing monoclonalantibody 24G9 was deposited with the American Type Culture Collection(ATCC), Rockville, Md., USA, under accession number HB 11780, and hasbeen accepted as a patent deposit, in accordance with the BudapestTreaty of 1977 governing the deposit of microorganisms for the purposesof patent procedure.

[0139] The hybridoma cell line 4A6(1)G1F7 producing monoclonal antibody4A6 was deposited with the American Type Culture Collection (ATCC),Rockville, Md., USA, under accession number HB 11943, and has beenaccepted as a patent deposit, in accordance with the Budapest Treaty of1977 governing the deposit of microorganisms for the purposes of patentprocedure.

[0140] The hybridoma cell line 5D3(1)F5D6 producing monoclonal antibody5D3 was deposited with the American Type Culture Collection (ATCC),Rockville, Md., USA, under accession number HB 11942, and has beenaccepted as a patent deposit, in accordance with the Budapest Treaty of1977 governing the deposit of microorganisms for the purposes of patentprocedure.

EXAMPLE 2 Assays

[0141] A. ELISA:

[0142] Individual wells of MaxiSorb™ immuno plates (Nunc, Naperville,Ill.) were coated with 0.2 ug IL-5 in 0.05M carbonate buffer pH 9.6.After incubating overnight at 4° C., the plates were rinsed with PBScontaining 0.025% Tween® 20, and blocked with 1% BSA in PBS with 0.025%Tween® 20 for two hours at room temperature. Undiluted hybridsupernatants were added to the IL-5 coated wells and incubated at roomtemperature for two hours. After the plates were rinsed, peroxidaselabeled goat anti-mouse IgG & IgM (Boehringer Mannheim, Indianapolis,Ind.) was added at 1/7500 dilution in PBS containing 1% BSA and 0.025%Tween® 20. Two hours later the plates were washed and 0.2 ml of 0.1Mcitrate buffer pH 4.75 containing 0.1% urea peroxide and 1 mg/mlorthophenylenediamine was added. After 15 min the plates were read at450 nm on a VMax™ Microplate Reader (Molecular Devices, Menlo Park,Calif.).

[0143] B. Receptor Binding Inhibition Assay:

[0144] Membrane extracts of Drosophila S2 cells expressing the α-chainof the human IL-5 Receptor (IL-5R) were used to measure the effect ofantibody on IL-5 binding to receptor. To prepare the membranes, 10⁹cells were pelleted at 1000×g at 4° C. for 10 min. The cell pellet wasfrozen in a dry ice/ethanol bath for 15 min. The pellet was thawed,resuspended in 10 ml PBS at 4° C. and pelleted at 1000×g for 10 min. Thecell pellet was washed 2× in PBS and resuspended in 13.5 ml Hypotonicbuffer (10 mM Tris pH 7.5, 3 mM MgCl₂, 1 mM dithiothreitol, 1 mMphenylmethylsulfonyl fluoride, 1 uM leupeptin, 1 uM pepstatin A) andincubated on ice for 5 min. The cell suspension was homogenized in a 15ml Dounce homogenizer and brought to a final concentration of 0.25 Msucrose with a solution of 2.5 M sucrose. Cell debris was removed by a15 min centrifugation at 1000×g. Cell membranes were pelleted at100,000×g at 4° C. for 90 min and resuspended in 50 ml of 10 mM Tris pH7.5, 3 mM MgCl₂, 250 mM sucrose, and stored at −70° C.

[0145] Assays with Drosophila membranes containing receptor wereperformed in MultiscreenGV™ plates (Millipore Corp., Bedford, Mass.)using Drosophila tissue culture medium M3 (Lindquist et al., DrosophilaInf. Serv., 58: 163 (1982)) containing 25 mM HEPES buffer pH 7.2 and0.1% BSA (Binding Buffer). Wells were pre-blocked with 0.1 ml bindingbuffer. 50 ul of the test sample, in triplicate, was added to wellsfollowed by 25 ul iodinated (¹²⁵I) IL-5. After 20 minutes incubation atroom temperature, 25 ul of the membrane extract of Drosophila S2 cellsexpressing the α-chain of the human IL5R was added to the wells. After 1hour further incubation the membranes were collected by vacuumfiltration and washed 3× with binding buffer. Filters were dried andcounted.

[0146] C. IL-5 Neutralization Assay:

[0147] The murine IL-5/IL-3 dependent cell line LyH7.B13 (B13) wasobtained courtesy of R. Palacios, Basel Institute of Immunology,Switzerland. Cells were subcultured twice weekly in RPMI 1640 medium(GibcoBRL, Renfrewshire, UK), supplemented with L-Glutamine,non-essential amino acids, sodium pyruvate, penicillin-streptomycin (allGibcoBRL), plus 2-mercaptoethanol (5×10⁻⁵ M, Sigma), 10% fetal bovineserum (Globepharm, Surrey, UK) and 1-16 units murine IL-5. For assays,cells were cultured for 48 hours in triplicate (5000 cells/well) in96-well round bottom plates in the presence of appropriately dilutedtest samples and pulsed with 0.5 uCi³H-thymidine (Amersham, Bucks, UK)for the final 4 hours. They were processed for scintillation counting ina 1205 Betaplate (LKB Wallac, Beds, UK).

[0148] D. Optical Biosensor:

[0149] Kinetic and equilibrium binding properties with immobilized hIL-5and antibodies were measured using a BIAcore optical biosensor(Pharmacia Biosensor, Uppsala, Sweden). Kinetic data were evaluatedusing relationships described previously (Karlsson et al., J. Immunol.Meth., 145:229-240 (1991)) and which is incorporated by reference in itsentirety.

[0150] Three of the neutralizing mAbs, namely 2B6, 2E3 and 2F2, had verysimilar potencies of inhibition of ¹²⁵I-IL-5 binding to membranereceptor and neutralization of B cell proliferation and also verysimilar affinities for IL-5 (see Table I). The nucleotide sequences ofthe V_(H) and V_(L) from these three mAbs, 2 IgG₁ and 1 IgG_(2a),respectively, were determined. The sequences obtained were very similar,differing only at a few residues. TABLE I Affinity and neutralizingactivity of mAbs reactive with human IL-5 Neutralization BindingProliferation 100% mAb Kd (pM)^(a) IC₅₀(nM)^(b) IC₅₀ ^(c) Inhibition^(c)2B6 22 1 70 200 2E3 20 1 90 600 2F2 13 1 150 340 1C6 86 43 12,200 ND24G9 ND >133 >100,000 ND 4A6 18 >88 28 100 5D3 ND ND 100 10,000

EXAMPLE 3 Isolation and Characterization of IL-5 Fabs from CombinatorialLibrary

[0151] A. PCR and Combinatorial Library Construction:

[0152] RNA purified from the spleens of three mice was reversetranscribed with a cDNA kit (Boehringer Mannheim, Indianapolis, Ind.)using either the primer (dT)15 supplied with the kit or the 3′Fd (IgG1,IgG2a & IgG3) and kappa light chain primers as described by Huse et al.(Science, 2:1275 (1989)) and Kang, S. A. (Methods: Companion MethodsEnzymol., 2:111 (1991)) which are hereby incorporated by reference intheir entirety. Immunoglobulin cDNAs were amplified by PCR using theprimers and the thermal cycling conditions described (Huse et al.supra). The Hot Start technique using AmpliWaxr PCR Gem 100 (PerkinElmer Cetus, Norwalk, Conn.) beads and the manufacturer's protocol wasused for all of the reactions. The PCR products were gel purified,digested, and ligated into the pMKFabGene3 vector (Ames et al., J.Immunol., 152:4572 (1994)). The library titer following ligation withthe Fd cDNAs was 5.1×10⁻¹¹ CFU and following ligation with the kappacDNAs was 1.5×10⁶ CFU. XL1-Blue cells (Stratagene, La Jolla, Calif.)transformed with the phagemid library were infected with helper phageVCSM13 (Stratagene) and phage were prepared as described by Barbas andLerner (Methods: Companion Methods Enzmmol., 2:119 (1991)).

[0153] B. Biopanning:

[0154] Four microtiter wells (Immulon II Removawell Strips, DynatechLaboratories Inc., Chantilly, Va.) were coated overnight at 4° C. withIL-5 (1 ug/well) in 0.1M bicarbonate, pH 8.6. The wells were washed withwater and blocked with PBS containing 3% BSA at 37° C. for 1 hour. Theblocking solution was removed, and the library was added to microtiterwells (50 ul/well) and incubated at 37° C. for 2 hours. Wells werewashed 10 times with TBS/Tween® (50mM Tris-HCl, pH 7.5, 150 mM NaCl,0.5% Tween® 20) and once with H₂O prior to elution of the adherent phagewith 0.1 M HCl, adjusted to pH 2.2 with glycine, containing 1 mg/ml BSA.

[0155] C. Colony Lifts:

[0156] Colony lifts from clones isolated from the third and fourthrounds of biopanning were processed as described (Barbas and Lerner,supra). Filters were incubated for 1 hour at room temperature with0.5-1.0 uCi ¹²⁵I-IL-5, which had been iodinated using Bolton-Hunterreagent (NEN, Billerica, Mass.) following the manufacturers recommendedprocedure, in PBS containing 1% BSA, washed with PBS 0.25% Tween, andexposed to Kodak XAR film. Colonies expressing IL-5-reactive Fabs weredetected by autoradiography.

[0157] D. Preparation of Soluble FABs:

[0158] Phagemid DNAs were digested with NheI and SpeI to remove gene IIIand self-ligated. XL1-Blue cells were transformed, and isolated cloneswere grown overnight at 37° C. in 5.0 ml super broth (SB) medium (30 gtryptone, 20 g yeast extract, 10 g 3-[N-Morpholino]propanesulfonic acid,MOPS with pH adjusted to 7) containing 1% glucose and 50 ug/mlcarbenicillin. Cells from 1 ml of this culture were pelleted at 3500 rpmfor 10 min in Beckman GS-6R centrifuge and used to inoculate 5 ml SBcontaining 50 ug/ml carbenicillin. Cultures were shaken for 1 hour at37° C., Isopopyl-b-D-thiogalactopyranoside (IPG; 1 mM) was added and thecultures were transferred to 28° C. overnight. Soluble Fab was preparedfrom periplasmic extracts by lysing the cell pellet for 20 min at 4° C.in 20% sucrose suspended in 30 mM Tris pH 8.0, followed bycentrifugation in a Microfuge for 10 min. Fab concentrations wereestimated by western blot by comparison to samples containing knownamounts of murine Fab. The different bacterial periplasmic extractscontained similar concentrations of Fab, ranging from 1 to 20 ug/ml, asestimated by western blot analysis.

[0159] E. Purification of FABs:

[0160] A chelating peptide was engineered onto the carboxy-terminal endof the heavy chain to aid in protein purification. Following removal ofthe M13 geneIII coding region, via digestion with NheI and SpeI, a pairof overlapping oligonucleotides: [SEQ ID NO: 43]5′-CTAGCCACCACCACCACCACCACTAA-3′; [SEQ ID NO: 44]3′-GGTGGTGGTGGTGGTGGTGATTGATC-5′ encoding six histidine residues weresubcloned into the Fab expression vector. Induction of Fab expressionwas performed as described above. Following overnight induction at 28°C. periplasmic lysate of the cell pellet was prepared by 30 minincubation at 4° C. in 20% sucrose, 30 mM TRIS pH 8.0. Urea and Brij-35detergent were added to the clarified supernatant to finalconcentrations of 2M and 1% respectively. After stirring at roomtemperature for 1 hour, the treated and clarified supernatant was loadedat 0.5 ml/min directly onto a 5 ml Nickel-NTA metal chelating column(1.5×3 cm) equilibrated with buffer A (100 mM Na-Phosphate, 10 mM Tris,0.3 M NaCl, 2 M urea, pH 8.0). After a 4 column volume (20 ml) washbound materials were eluted with a 6 column volume (30 ml) reverse pHgradient from pH 8 to pH 4 in the same buffer as above. The purifiedFabs eluted from the column in a sharp symmetrical peak at pH 5.5. Theywere >90% pure and free of DNA.

[0161] F. FAB ELISA:

[0162] Immulon II plates (Dynatech) were coated overnight at 4° C. withprotein suspended (1 mg/ml; 50 ml per well) in 0.1 M bicarbonate buffer,pH 8.6. Dilutions and washes were performed in PBS containing 0.05%Tween™ 20. Plates were washed and blocked for 1 hour with PBS containing1% BSA at room temperature. Various dilutions of the bacterialsupernatants containing soluble Fabs, or purified Fabs, were added tothe plates. Following a one hour incubation plates were washed andbiotinylated goat anti-mouse kappa (Southern Biotechnology Associates,Inc., Birmingham, Ala.) was added (1:2000 dilution; 50 ul/well) for 1hour. The plates were washed and streptavidin labeled horseradishperoxidase was added (1:2000 dilution; 50 ul/well) for 1 hour. Theplates were washed, ABTS peroxidase substrate was added (100 ul/well;Kirkegaard & Perry Laboratories, Gaithersburg, Md.) and the opticaldensity at 405 nm was read on a UVmax™ (Molecular Devices) microplatereader.

[0163] G. Isolation and Characterization of Fabs from a CombinatorialLibrary:

[0164] Phage bearing Fabs to IL-5 were selected from the library bymultiple rounds of biopanning against microtiter wells coated with IL-5.After 4 rounds of selection IL-5 reactive Fabs were identified by acolony lift assay using ¹²⁵I-IL-5. Thirty four colonies from the thirdround and 4 colonies from the fourth round were identified which boundlabeled IL-5. Binding to IL-5 was confirmed by direct binding ELISAusing culture supernatants expressing the Fab-geneIII fusion protein.DNA was isolated from these colonies and, after removing the codingregion of M13 gene III, soluble Fab expression was induced. Periplasmicfractions were prepared and assayed by ELISA for binding to IL-5. TheFabs bound specifically to IL-5 with no demonstrable binding to ananother protein, rC5a.

[0165] The undiluted periplasmic extracts (containing 1 to 20 ug/ml Fab)were assayed in the IL-5R binding inhibition assay (Example 2). None ofthe Fabs inhibited binding of iodinated IL-5 to the IL-5Rα by more than35%.

[0166] H. Conversion of Neutralizing mAb to a FAB:

[0167] The Fd and κ cDNAs of mAb (2B6) were isolated by PCR using theconditions described above. The gel-purified fragments were subclonedinto the pMKFabGene3 vector which had been modified to include thehexa-His sequence 3′ of the gene III cDNA, resulting in the plasmidpMKFabGene3H. A functional, IL-5 binding Fab clone containing the 2B6heavy and light chains was identified by a colony lift assay. Uponremoval gene III via Nhe I/SpeI I digestion and self-ligation the heavychain was fused in frame to the hexa-His, allowing purification asdescribed above. In a dose dependent manner, this Fab inhibited receptorbinding with an IC₅₀ of approximately 7.5 ug/ml, similar to that of theparent mAb, murine 2B6.

[0168] I. Construction and Screening of Chain-Shuffled Library:

[0169] The cDNA encoding the Fd of the neutralizing mAb 2B6 wassubcloned as an XhoI/SpeI fragment into pMKFabGene3H which contained aSstI/XbaI fragment in lieu of a light chain cDNA. This phagemid wasdigested with SstI and XbaI and ligated with the SstI/XbaI digestedlight chain PCR product derived from the IL-5 immunized mice (describedabove). The library titer following ligation was 4×10⁵ CFU. Biopanning,and colony lift assay was performed as described above for thecombinatorial library.

[0170] The library was constructed by pairing the cDNA encoding the Fdof the neutralizing mAb 2B6 with the same light chain repertoire,recovered from the IL-5 immunized mice, used to generate thecombinatorial library. This chain shuffled library was subjected to 4rounds of biopanning vs immobilized IL-5 and the resultant colonies wereassayed for IL-5 reactivity using the colony lift assay. Positivecolonies, which bound iodinated IL-5, were further assayed by ELISA andthe IL-5Rα binding assay. Two of the Fabs, 2 & 15, recovered from thechain shuffled library blocked binding of IL-5 to the IL-5Rα andinhibited IL-5 dependent proliferation in the B13 assay. The sequencesof these 2 Vks were similar to the sequence of the 2B6 Vk, the originallight chain partner for the 2B6 V_(H). The light chain sequences for Fab2 & 15 are SEQ D NOs: 45 and 46, respectively. For Fab 2, CDRs 1-3 areSEQ ID NOs: 10, 11 and 47, respectively. For Fab 15, CDRs 1-3 are SEQ IDNOs: 10, 11 and 48, respectively.

[0171] All antibody amino acid sequences listed below in Examples 4 and5 use the KABAT numbering system which allows variability in CDR andframework lengths. That is, key amino acids are always assigned the samenumber regardless of the actual number of amino acids preceding them.For example, the cysteine preceding CDR1 of all light chains is alwaysKABAT position 23 and the tryptophan residue following CDR1 is alwaysKABAT position 35 even though CDR1 may contain up to 17 amino acids.

EXAMPLE 4 Humanized Antibody

[0172] One humanized antibody was designed to contain murine CDRs withina human antibody framework. This humanized version of the IL-5 specificmouse antibody 2B6, was prepared by performing the followingmanipulations.

[0173] A. Gene Cloning:

[0174] mRNA was isolated from each of the respective 2B6, 2F2 and 2E3hybridoma cell lines (see Example 1) with a kit obtained from BoehringerMannheim (Indianapolis, Ind.) and then reverse transcribed using theprimer (dT)15 supplied with a cDNA kit (Boehringer Mannheim) to makecDNA. PCR primers specific for mouse immunoglobulin were used to amplifyDNA coding for domains extending from amino acid #9 (KABAT numberingsystem) of the heavy chain variable region to the hinge region and fromamino acid #9 (KABAT numbering system) of the light chain variableregion to the end of the constant region. Several clones of eachantibody chain were obtained by independent PCR reactions.

[0175] The mouse gamma 1 hinge region primer used is [SEQ ID NO: 22]:

[0176] 5′ GTACATATGCAAGGCTTACAACCACAATC 3′.

[0177] The mouse gamma 2a hinge region primer used is [SEQ ID NO: 23]:

[0178] 5′ GGACAGGGCTTACTAGTGGGCCCTCTGGGCTC 3′

[0179] The mouse heavy chain variable region primer used is [SEQ ID NO:24]:

[0180] 5′ AGGT(C or G)(C or A)A(G or A)CT(G or T)TCTCGAGTC(T or A)GG 3′

[0181] The mouse kappa chain constant region primer used is [SEQ ID NO:25]:

[0182] 5′ CTAACACTCATTCCTGTTGAAGCTCTTGACAATGGG 3′

[0183] The mouse light chain variable region primer is [SEQ ID NO: 26]:

[0184] 5′ CCAGATGTGAGCTCGTGATGACCCAGACTCCA 3′

[0185] The PCR fragments were cloned into plasmids pGEM7f+(Promega) thatwere then transformed into E. coli DH5a (Bethesda Research Labs).

[0186] B. DNA Sequencing:

[0187] The heavy and light chain murine cDNA clones from Part A abovewere sequenced. The results of sequencing of the variable regions ofthese clones are shown in SEQ ID NOs: 1-6 (FIG. 1-6). Each clonecontained amino acids known to be conserved among mouse heavy chainvariable regions or light chain variable regions. The CDR amino acidsequences are listed below.

[0188] The CDR regions for the 2B6 heavy chain are SEQ ID NOs: 7, 8 and9. See FIG. 7. These sequences are encoded by SEQ ID NO: 1. The CDRregions for the light chain are SEQ ID NOs: 10, 11 and 12. See FIG. 7.These sequences are encoded by SEQ ID NO:2.

[0189] The CDR regions for the 2F2 heavy chain are SEQ ID NOs: 7, 8 and9. See FIG. 7. These sequences are encoded by SEQ ID NO:3. The CDRregions for the light chain are SEQ ID NOs: 10, 11 and 13. See FIG. 7.These sequences are encoded by SEQ ID NO:4.

[0190] The CDR regions for the 2E3 heavy chain are SEQ ID NOs: 7, 8 and14. See FIG. 7. These sequences are encoded by SEQ ID NO:5. The CDRregions for the light chain are SEQ ID NOs: 10, 11 and 13. See FIG. 7.These sequences are encoded by SEQ ID NO:6.

[0191] C. Selection of Human Frameworks:

[0192] Following the cloning of 2B6, the amino acid sequences of thevariable region heavy and light chains (FIGS. 1 and 2) (SEQ ID NOs: 15and 16, respectively) were compared with the known murine immunoglobulinsequences in the KABAT and SWISS-PROT (Nuc. Acids Res., 20:2019-2022(1992)) protein sequence databases in order to assign amino acids to theN-terminal residues. The 2B6 heavy and light chain variable regiondeduced amino acid sequences were then compared with the humanimmunoglobulin protein sequence databases in order to identify a humanframework for both the heavy and light chains which would most closelymatch the murine sequence. In addition, the heavy and light chains wereevaluated with a positional database generated from structural models ofthe Fab domain to assess potential conflicts due to amino acids whichmight influence CDR presentation. Conflicts were resolved duringsynthesis of the humanized variable region frameworks by substitution ofthe corresponding mouse amino acid at that location.

[0193] The heavy chain framework regions of an antibody obtained from ahuman myeloma immunoglobulin (COR) was used (E. M. Press and N. M. Hogg,Biochem. J., 117:641-660 (1970)). The human heavy chain framework aminoacid sequence was found to be approximately 66% homologous to the 2B6framework.

[0194] For a suitable light chain variable region framework, the lightchain variable framework sequence of the Bence-Jones protein, (LEN)(Schneider et al., Hoppe-Seyler's Z. Phvsiol. Chem., 3:507-557 (1975)),was used. The human light chain framework regions were approximately 82%homologous to the murine 2B6 light chain framework regions, at the aminoacid level.

[0195] The selected human frameworks were back translated to provide aDNA sequence.

[0196] D. Construction of Humanized MAb Genes:

[0197] Given the 2B6 heavy chain CDRs [FIG. 7 and SEQ ID NOs: 1-2] andthe framework sequences of the human antibodies, a synthetic heavy chainvariable region was made [SEQ ID NO: 18]. This was made using foursynthetic oligonucleotides [SEQ ID NOs:27 and 28] [SEQ ID NOs: 29 and30] which, when joined, coded for amino acids #21-#106 (KABATnumeration). The oligonucleotides were then ligated into the HpaI-KpnIrestriction sites of a pUC18 based plasmid containing sequences derivedfrom another humanized heavy chain based on the COR framework (supra).This plasmid provides a signal sequence [SEQ ID NO: 17] and theremaining variable region sequence. Any errors in the mapped sequencewere corrected by PCR with mutagenic primers or by the addition ofsynthetic linkers into existing restriction sites.

[0198] The signal sequence and humanized heavy chain variable regionwere excised from the pUC based plasmid as a EcoRI-ApaI fragment andligated into the expression vector pCD that contained an IgG₁ humanconstant region. The synthetic heavy chain variable region nucleotideand amino acid sequences are provided in FIG. 8 [SEQ ID NOs: 18 and 19].The human framework residues are amino acids 1-30, 36-49, 66-97 and109-119 of SEQ ID NO: 19. The amino acid sequences of the CDRs areidentical to the murine 2B6 CDRs. The resulting expression vector,pCDIL5HZHC1.0, is shown in FIG. 10.

[0199] Given the 2B6 light chain CDRs [FIG. 7 and SEQ ID NOs: 10, 11 and12] and the framework sequence of the human antibody, a synthetic lightchain variable region was made [SEQ ID NO: 20]. Four syntheticoligonucleotides coding for amino acids #27-#58 (KABAT numeration)[SEQID NOs:31 and 32] and amino acids #80-#109 [SEQ ID NOs:33 and 34] of thehumanized V_(L) with SacI-KpnI and PstI-HindIII ends respectively, wereinserted into a pUC18 based plasmid containing sequences derived fromanother human light chain framework (B17) (Marsh et al, Nuc, Acids Res,13:6531-6544 (1985)) which shares a high degree of homology to the LENframework. This plasmid provides the remaining variable region sequence.Any errors in the mapped sequence and the single amino acid differencebetween the LEN and B17 frameworks were corrected by PCR with mutagenicprimers or by the addition of synthetic linkers into existingrestriction sites.

[0200] The humanized light chain variable region was isolated from thepUC plasmid as a EcoRV-NarI fragment and ligated into the expressionvector pCN that contained a signal sequence [SEQ ID NO: 17] along with akappa human constant region. The synthetic light chain variable regionnucleotide and amino acid sequences are provided in FIG. 9 [SEQ IDNOs:20 and 21]. The human framework residues are amino acids 1-23,41-55, 63-94 and 104-113 of SEQ ID NO: 21. The amino acid sequences ofthe CDRs are identical to the murine 2B6 CDRs. However, the codingsequences for these CDRs differ from the murine 2B6 coding sequences toallow creation of restriction enzyme sites. One of the resultingexpression vectors, pCNIL5HZLC1.0, is shown in FIG. 11. These syntheticvariable light and/or heavy chain sequences are employed in theconstruction of a humanized antibody.

[0201] E. Expression of Humanized MAb:

[0202] The humanized heavy chain, derived from an IgG₁ isotype, utilizesa synthetic heavy chain variable region as provided in SEQ ID NO: 19.This synthetic V_(H) containing the 2B6 heavy chain CDRs was designedand synthesized as described above.

[0203] The humanized light chain, a human kappa chain, utilizes asynthetic light chain variable region as provided in SEQ ID NO: 21. Thissynthetic V_(L) containing the 2B6 light chain CDRs was designed andsynthesized as described above. The DNA fragments coding for thehumanized variable regions were inserted into pUC19-based mammalian cellexpression plasmids that utilize a signal sequence and contain CMVpromoters and the human heavy chain or human light chain constantregions of the chimera produced in Example 5 below, by conventionalmethods (Maniatis et al., cited above) to yield the plasmidspCDIL5HZHC1.0 (heavy chain) [SEQ ID NO: 49, see also FIG. 10] andpCNIL5HZLC1.0 (light chain) [SEQ ID NO: 50, see also FIG. 11]. Theplasmids were co-transfected into COS cells and supernatants assayedafter three and five days, respectively, by the ELISA described inExample 5 for the presence of human antibody.

[0204] The above example describes the preparation of an exemplaryengineered antibody. Similar procedures may be followed for thedevelopment of other engineered antibodies, using other anti-IL-5antibodies (e.g., 2F2, 2E3, 4A6, 5D3, 24G9, etc.) developed byconventional means.

[0205] F. Purification:

[0206] Purification of CHO expressed chimeric and humanized 2B6 can beachieved by conventional protein A (or G) affinity chromatographyfollowed by ion exchange and molecular sieve chromatography. Similarprocesses have been successfully employed for the purification to >95%purity of other mAbs (e.g., to respiratory syncytial virus,interleukin-4 and malaria circumsporozoite antigens).

[0207] G. Additional Humanized mAbs and Expression Plasmids:

[0208] Given the plasmid pCDIL5HZHC1.0 [SEQ ID NO: 49] the expressionplasmid pCDIL5HZHC1.1 was made that substitutes an Asparagine forThreonine at framework position 73. This was done by ligating asynthetic linker with EcoRV and XhoI ends [SEQ ID NO: 51 and SEQ ID NO:52] into identically digested pCDIL5HZHC1.0. Similarly, the expressionplasmid pCDIL5HZHC1.2 substitutes an Isoleucine for Valine at frameworkposition 37. This was accomplished by ligating a synthetic linker withHpaI and XbaI ends [SEQ ID NO: 53 and SEQ ID NO: 54] into identicallydigested pCDIL5HZHC1.0. The expression plasmid pCDIL5HZHC1.3 was alsomade by ligating a synthetic linker with HpaI and XbaI ends [SEQ ID NO:53 and SEQ ID NO: 54] into identically digested pCDIL5HZHC1.1

[0209] Given the pUC18 based plasmid described previously which containsDNA sequences of four synthetic oligonucleotides [SEQ ID NOs: 31, 32, 33and 34], a humanized light chain variable region was made whereframework position #15 is changed from a Leucine to Alanine. Thisplasmid was digested with NheI and SacI restriction endonucleases and asynthetic linker [SEQ ID NOs: 55 and 56] was inserted. An EcoRV-NarIfragment was then isolated and ligated into the identically digestedexpression vector pCNIL5HZLC1.0 to create pCNIL5HZLC1.1.

[0210] A synthetic variable region was made using the heavy chainframework regions obtained from immunoglobulin (NEW) (Saul et al, J.Biol. Chem. 253:585-597(1978)) and the 2B6 heavy chain CDRs [FIG. 7 andSEQ ID NOs: 1-2]. Framework amino acids which might influence CDRpresentation were identified and substitutions made using methodsdescribed previously. Four overlapping synthetic oligonucleotides weregenerated [SEQ ID NOs: 57, 58, 59 and 60] which, when annealed andextended, code for amino acids representing a signal sequence [SEQ IDNO: 17] and a heavy chain variable region. This synthetic gene was thenamplified using PCR primers [SEQ ID NOs: 63 and 64] and ligated as aBstXI-HindIII restriction fragment into a pUC18 based plasmid containingsequences derived from another humanized heavy chain based on the CORframework. A phenylalanine to tyrosine framework substitution was madeat amino acid position 91 (Kabat numbering system) (equivalent toposition 94 of FIG. 12) by inserting a synthetic oligonucleotide linker[SEQ ID NOs: 75 and 76] into SacII and KpnI restriction sites. Theresulting heavy chain variable region [FIG. 12 and SEQ ID NOs: 61, 62]is referred to as the NEWM humanized heavy chain.

[0211] Any errors in the mapped sequence were corrected by PCR withmutagenic primers or by the addition of synthetic linkers into existingrestriction sites. The signal sequence and humanized heavy chainvariable region were excised from the pUC based plasmid as a EcoRI-ApaIfragment and ligated into the expression vector pCD that contained ahuman IgG₁ constant region to create the plasmid pCDIL5NEWM. The aminoacid sequences of the CDRs are identical to the murine 2B6 heavy chainCDRs.

[0212] A synthetic variable region was made using the light chainframework regions obtained from immunoglobulin (REI) (Palm et al,Hoppe-Sevler's Z. Physiol. Chem. 3:167-191(1975)) and the 2B6 lightchain CDRs [FIG. 7 and SEQ ID NOs: 10, 11 and 12]. Framework amino acidswhich might influence CDR presentation were identified and substitutionsmade using methods described previously. Four overlapping syntheticoligonucleotides were generated [SEQ ID NOs: 65, 66, 67 and 68] which,when annealed and extended, code for amino acids representing a lightchain variable region [FIG. 13 and SEQ ID NOs: 69, 70] referred to asthe REI humanized light chain. This synthetic gene was then amplifiedusing PCR primers [SEQ ID NOs: 71 and 72] and ligated as anEcoRI-HindIII restriction fragment into pGEM-7Zf(+) (PromegaCorporation, Madison, Wis.).

[0213] Any errors in the mapped sequence were corrected by PCR withmutagenic primers or by the addition of synthetic linkers into existingrestriction sites. The humanized light chain variable region was excisedfrom the pGEM-7Zf(+) based plasmid as an EcoRV-NarI fragment and ligatedinto the expression vector pCN that contained a signal sequence [SEQ IDNO: 17] along with a human Kappa constant region to create the plasmidpCNIL5REI. The amino acid sequences of the CDRs are identical to themurine 2B6 light chain CDRs. However, the coding sequences for theseCDRs differ from the murine 2B6 coding sequences to allow creation ofrestriction enzyme sites. These synthetic variable light and/or heavychain sequences are employed in the construction of a humanizedantibody.

[0214] Given the pGEM-7Zf(+) based plasmid described above, a humanizedlight chain variable region can be made where framework position #15 ischanged from a Valine to Alanine. This plasmid may be digested with NheIand SacI restriction endonucleases and a synthetic linker [SEQ ID NOs:73 and 74] is inserted. An EcoRV-NarI fragment may then be isolated andligated into the identically digested expression vector pCNIL5HZREI tocreate the plasmid pCNIL5REI_(VI5A).

EXAMPLE 5 Construction of a Chimeric Antibody

[0215] DNA coding for amino acids #9-#104 (KABAT numeration) of themurine mAb 2B6 heavy chain variable region was isolated as a AvaII-StyIrestriction fragment from a pGEM7Zf+based PCR clone of cDNA generatedfrom the 2B6 hybridoma cell line (see Example 4). The flanking heavychain variable region sequences and a signal sequence [SEQ ID NO: 17]were provided by combining this fragment along with four small syntheticoligomer linkers [SEQ ID NOs: 35 and 36] [SEQ ID NOs: 37 and 38] into apUC18 based plasmid digested with BstXI-HindIII. A consensus ofN-terminal amino acids deduced from closely related murine heavy chainswere assigned for the first eight V_(H) residues and are coded withinSEQ ID NOs: 35 and 36. The deduced amino acid sequence of the heavychain was verified by the sequencing of the first 15 N-terminal aminoacids of the 2B6 heavy chain.

[0216] An EcoRI-ApaI fragment containing sequence for signal and V_(H)regions was isolated and ligated into plasmid pCD that already encodesthe human IgG₁ constant region.

[0217] DNA coding for amino acids #12-#99 (KABAT nomenclature) of themurine mAb 2B6 light chain variable region was isolated as a DdeI-AvaIrestriction fragment from a pGEM7Zf+based PCR clone of cDNA generatedfrom the 2B6 hybridoma cell line (see Example 4). The flanking lightchain variable region sequences were provided by combining this fragmentalong with four small synthetic oligomer linkers [SEQ ID NOs: 39 and 40][SEQ ID NOs: 41 and 42] into a pUC18 based plasmid digested withEcoRV-HindIII. A consensus of N-terminal amino acids deduced fromclosely related murine light chains were assigned for the first eightV_(L) residues and are coded within SEQ ID NOs: 39 and 40. The deducedamino acid sequence of the light chain was verified by the sequencing ofthe first 15 N-terminal amino acids of the 2B6 light chain. Thisvariable region was then isolated as a EcoRV-NarI fragment and ligatedinto the expression vector pCN that already contains the human kapparegion and a signal sequence.

[0218] Expression of a chimeric antibody was accomplished byco-transfection of the pCD and pCN based plasmids into COS cells.Culture supernatants were collected three and five days later andassayed for immunoglobulin expression by ELISA described as follows:Each step except for the last is followed by PBS washes. Microtiterplates were coated overnight with 100 ng/50 ul/well of a goat antibodyspecific for the Fc region of human antibodies. The culture supernatantswere added and incubated for 1 hour. Horseradish peroxidase conjugatedgoat anti-human IgG antibody was then added and allowed to incubate for1 hour. This was followed by addition of ABTS peroxidase substrate(Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.). After 1 hourincubation, the absorbance at 405 nm was read on a microtiter platereader (Molecular Devices Corporation, Menlo Park, Calif.). Expressionof the chimeric antibody was detected. In a similar ELISA, the COS cellsupernatants, containing the chimeric antibody, bound specifically tomicrotiter wells coated with human IL-5 protein. This result confirmedthat genes coding for an antibody to IL-5 had been synthesized andexpressed.

[0219] The above example describes the preparation of an exemplaryengineered antibody. Similar procedures may be followed for thedevelopment of other engineered antibodies, using other anti-IL-5 donorantibodies (e.g., 2F2, 2E3, 4A6, 5D3, 24G9, etc.) developed byconventional means.

1 76 334 base pairs nucleic acid single linear DNA (genomic)misc_feature 1..334 /note= “First base corresponds to Kabat position 24”1 ACCTGGCCTG GTGGCGCCCT CACAGAGCCT GTCCATCACT TGCACTGTCT CTGGGTTTTC 60ATTAACCAGC TATAGTGTAC ACTGGGTTCG CCAGCCTCCA GGAAAGGGTC TGGAGTGGCT 120GGGAGTAATA TGGGCTAGTG GAGGCACAGA TTATAATTCG GCTCTCATGT CCAGACTGAG 180CATCAGCAAA GACAACTCCA AGAGCCAAGT TTTCTTAAAA CTGAACAGTC TGCAAACTGA 240TGACACAGCC ATGTACTACT GTGCCAGAGA TCCCCCTTCT TCCTTACTAC GGCTTGACTA 300CTGGGGCCAA GGCACCACTC TCACAGTCTC CTCA 334 315 base pairs nucleic acidsingle linear DNA (genomic) misc_feature 1..315 /note= “First basecorresponds to Kabat position 25” 2 TCCTCCCTGA GTGTGTCAGC AGGAGAGAAGGTCACTATGA GCTGCAAGTC CAGTCAGAGT 60 CTGTTAAACA GTGGAAATCA AAAGAACTACTTGGCCTGGT ACCAGCAGAA ACCAGGGCAG 120 CCTCCTAAAC TTTTGATCTA CGGGGCATCCACTAGGGAAT CTGGGGTCCC TGATCGCTTC 180 ACAGGCAGTG GATCTGGAAC CGATTTCACTCTTTCCATCA GCAGTGTGCA GGCTGAAGAC 240 CTGGCAGTTT ATTACTGTCA GAATGTTCATAGTTTTCCAT TCACGTTCGG CTCGGGGACA 300 GAGTTGGAAA TAAAA 315 334 base pairsnucleic acid single linear DNA (genomic) misc_feature 1..334 /note=“First base corresponds to Kabat position 24” 3 ACCTGGCCTG GTGGCGCCCTCACAGAGCCT GTCCATCACT TGCACTGTCT CTGGGTTTTC 60 ATTAACCAGT TATAGTGTACACTGGGTTCG CCAGCCTCCA GGAAAGGGTC TGGAGTGGCT 120 GGGAGTAATA TGGGCTAGTGGAGGCACAGA TTATAATTCG GCTCTCATGT CCAGACTGAG 180 CATCAGCAAA GACAACTCCAAGAGCCAAGT TTTCTTAAAA CTGAACAGTC TGCGAACTGA 240 TGACACAGCC ATGTACTACTGTGCCAGAGA TCCCCCTTCT TCCTTACTAC GGCTTGACTA 300 CTGGGGCCAA GGCACCACTCTCACAGTCTC CTCA 334 315 base pairs nucleic acid single linear DNA(genomic) misc_feature 1..315 /note= “First base corresponds to Kabat25” 4 TCCTCCCTGA GTGTGTCAGC AGGAGAGAAG GTCACTATGA GCTGCAAGTC CAGTCAGAGT60 CTATTAAACA GTGGAAATCA AAAGAACTAC TTGGCCTGGT ACCAACAGAA ACCAGGGCAG 120CCTCCTAAAC TTTTGATCTA CGGGGCATCC ACTAGGGAAT CTGGGGTCCC TGATCGCTTC 180ACAGGCAGTG GATCTGGAAC CGATTTCACT CTTACCATCA GCAGTGTGCA GGCTGAAGAC 240CTGGCAGTTT ATTACTGTCA GAATGATCAT AGTTTTCCAT TCACGTTCGG CTCGGGGACA 300GAGTTGGAAA TAAAA 315 334 base pairs nucleic acid single linear DNA(genomic) misc_feature 1..334 /note= “First base corresponds to Kabatposition 24” 5 ACCTGGCCTG GTGGCGCCCT CACAGAGCCT GTCCATCACT TGCACTGTCTCTGGGTTTTC 60 ATTAACCAGC TATAGTGTAC ACTGGGTTCG CCAGCCTCCA GGAAAGGGTCTGGAGTGGCT 120 GGGAGTAATC TGGGCTAGTG GAGGCACAGA TTATAATTCG GCTCTCATGTCCAGACTGAG 180 CATCAGCAAA GACAACTCCA AGAGCCAAGT TTTCTTAAAA CTGAACAGTCTGCAAACTGA 240 TGACGCAGCC ATGTACTACT GTGCCAGAGA TCCCCCTTTT TCCTTACTACGGCTTGACTT 300 CTGGGGCCAA GGCACCACTC TCACAGTCTC CTCA 334 315 base pairsnucleic acid single linear DNA (genomic) misc_feature 1..315 /note=“First base corresponds to Kabat position 25” 6 TCCTCTCTGA GTGTGTCAGCAGGAGAGAAG GTCACTATGA GCTGCAAGTC CAGTCAGAGT 60 CTGTTAAACA GTGGAAATCAAAAAAACTAC TTGGCCTGGT ACCAGCAGAA ACCAGGGCAG 120 CCTCCTAAAC TTTTGATCTACGGGGCATCC ACTAGGGAAT CTGGGGTCCC TGATCGCTTC 180 ACAGGCAGTG GATCTGGAACCGATTTCACT CTTACCATCA GCAGTGTGCA GGCTGAAGAC 240 CTGGCAGTTT ATTACTGTCAGAATGATCAT AGTTTTCCAT TCACGTTCGG CTCGGGGACA 300 GAGTTGGAAA TAAAA 315 5amino acids amino acid single linear protein 7 Ser Tyr Ser Val His 1 516 amino acids amino acid single linear protein 8 Val Ile Trp Ala SerGly Gly Thr Asp Tyr Asn Ser Ala Leu Met Ser 1 5 10 15 11 amino acidsamino acid single linear protein 9 Asp Pro Pro Ser Ser Leu Leu Arg LeuAsp Tyr 1 5 10 17 amino acids amino acid single linear protein 10 LysSer Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15Ala 7 amino acids amino acid single linear protein 11 Gly Ala Ser ThrArg Glu Ser 1 5 9 amino acids amino acid single linear protein 12 GlnAsn Val His Ser Phe Pro Phe Thr 1 5 9 amino acids amino acid singlelinear protein 13 Gln Asn Asp His Ser Phe Pro Phe Thr 1 5 11 amino acidsamino acid single linear protein 14 Asp Pro Pro Phe Ser Leu Leu Arg LeuAsp Phe 1 5 10 119 amino acids amino acid single linear protein 15 GlnVal Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Ser Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ala Ser Gly Gly Thr Asp Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 7580 Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 9095 Arg Asp Pro Pro Ser Ser Leu Leu Arg Leu Asp Tyr Trp Gly Gln Gly 100105 110 Thr Thr Leu Thr Val Ser Ser 115 113 amino acids amino acidsingle linear protein 16 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu SerVal Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser GlnSer Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr GlnGln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser ThrArg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly ThrAsp Phe Thr Leu Ser 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu AlaVal Tyr Tyr Cys Gln Asn 85 90 95 Val His Ser Phe Pro Phe Thr Phe Gly SerGly Thr Glu Leu Glu Ile 100 105 110 Lys 60 base pairs nucleic acidsingle linear DNA (genomic) 17 ATGGTGTTGC AGACCCAGGT CTTCATTTCTCTGTTGCTCT GGATCTCTGG TGCCTACGGG 60 357 base pairs nucleic acid singlelinear DNA (genomic) 18 CAGGTTACCC TGCGTGAATC CGGTCCGGCA CTAGTTAAACCGACCCAGAC CCTGACGTTA 60 ACCTGCACCG TCTCCGGTTT CTCCCTGACG AGCTATAGTGTACACTGGGT CCGTCAGCCG 120 CCGGGTAAAG GTCTAGAATG GCTGGGTGTA ATATGGGCTAGTGGAGGCAC AGATTATAAT 180 TCGGCTCTCA TGTCCCGTCT GTCGATATCC AAAGACACCTCCCGTAACCA GGTTGTTCTG 240 ACCATGACTA ACATGGACCC GGTTGACACC GCTACCTACTACTGCGCTCG AGATCCCCCT 300 TCTTCCTTAC TACGGCTTGA CTACTGGGGT CGTGGTACCCCAGTTACCGT GAGCTCA 357 119 amino acids amino acid single linear protein19 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 510 15 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 2025 30 Ser Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 3540 45 Gly Val Ile Trp Ala Ser Gly Gly Thr Asp Tyr Asn Ser Ala Leu Met 5055 60 Ser Arg Leu Ser Ile Ser Lys Asp Thr Ser Arg Asn Gln Val Val Leu 6570 75 80 Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala85 90 95 Arg Asp Pro Pro Ser Ser Leu Leu Arg Leu Asp Tyr Trp Gly Arg Gly100 105 110 Thr Pro Val Thr Val Ser Ser 115 339 base pairs nucleic acidsingle linear DNA (genomic) 20 GATATCGTGA TGACCCAGTC TCCAGACTCGCTAGCTGTGT CTCTGGGCGA GAGGGCCACC 60 ATCAACTGCA AGAGCTCTCA GAGTCTGTTAAACAGTGGAA ATCAAAAGAA CTACTTGGCC 120 TGGTATCAGC AGAAACCCGG GCAGCCTCCTAAGTTGCTCA TTTACGGGGC GTCGACTAGG 180 GAATCTGGGG TACCTGACCG ATTCAGTGGCAGCGGGTCTG GGACAGATTT CACTCTCACC 240 ATCAGCAGCC TGCAGGCTGA AGATGTGGCAGTATACTACT GTCAGAATGT TCATAGTTTT 300 CCATTCACGT TCGGCGGAGG GACCAAGTTGGAGATCAAA 339 113 amino acids amino acid single linear protein 21 AspIle Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 7580 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 9095 Val His Ser Phe Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100105 110 Lys 29 base pairs nucleic acid single linear DNA (genomic) 22GTACATATGC AAGGCTTACA ACCACAATC 29 32 base pairs nucleic acid singlelinear DNA (genomic) 23 GGACAGGGCT TACTAGTGGG CCCTCTGGGC TC 32 23 basepairs nucleic acid single linear DNA (genomic) 24 AGGTSMARCT KTCTCGAGTCWGG 23 36 base pairs nucleic acid single linear DNA (genomic) 25CTAACACTCA TTCCTGTTGA AGCTCTTGAC AATGGG 36 32 base pairs nucleic acidsingle linear DNA (genomic) 26 CCAGATGTGA GCTCGTGATG ACCCAGACTC CA 32140 base pairs nucleic acid single linear DNA (genomic) 27 AACCTGCACCGTCTCCGGTT TCTCCCTGAC GAGCTATAGT GTACACTGGG TCCGTCAGCC 60 GCCGGGTAAAGGTCTAGAAT GGCTGGGTGT AATATGGGCT AGTGGAGGCA CAGATTATAA 120 TTCGGCTCTCATGTCCCGTC 140 149 base pairs nucleic acid single linear DNA (genomic)28 ATATCGACAG ACGGGACATG AGAGCCGAAT TATAATCTGT GCCTCCACTA GCCCATATTA 60CACCCAGCCA TTCTAGACCT TTACCCGGCG GCTGACGGAC CCAGTGTACA CTATAGCTCG 120TCAGGGAGAA ACCGGAGACG GTGCAGGTT 149 139 base pairs nucleic acid singlelinear DNA (genomic) 29 TGTCGATATC CAAAGACACC TCCCGTAACC AGGTTGTTCTGACCATGACT AACATGGACC 60 CGGTTGACAC CGCTACCTAC TACTGCGCTC GAGATCCCCCTTCTTCCTTA CTACGGCTTG 120 ACTACTGGGG TCGTGGTAC 139 126 base pairsnucleic acid single linear DNA (genomic) 30 CACGACCCCA GTAGTCAAGCCGTAGTAAGG AAGAAGGGGG ATCTCGAGCG CAGTAGTAGG 60 TAGCGGTGTC AACCGGGTCCATGTTAGTCA TGGTCAGAAC AACCTGGTTA CGGGAGGTGT 120 CTTTGG 126 117 basepairs nucleic acid single linear DNA (genomic) 31 CTCAGAGTCT GTTAAACAGTGGAAATCAAA AGAACTACTT GGCCTGGTAT CAGCAGAAAC 60 CCGGGCAGCC TCCTAAGTTGCTCATTTACG GGGCGTCGAC TAGGGAATCT GGGGTAC 117 117 base pairs nucleic acidsingle linear DNA (genomic) 32 CCCAGATTCC CTAGTCGACG CCCCGTAAATGAGCAACTTA GGAGGCTGCC CGGGTTTCTG 60 CTGATACCAG GCCAAGTAGT TCTTTTGATTTCCACTGTTT AACAGACTCT GAGAGCT 117 102 base pairs nucleic acid singlelinear DNA (genomic) 33 GCTGAAGATG TGGCAGTATA CTACTGTCAG AATGTTCATAGTTTTCCATT CACGTTCGGC 60 GGAGGGACCA AGTTGGAGAT CAAACGTACT GTGGCGGCGC CA102 111 base pairs nucleic acid single linear DNA (genomic) 34AGCTTGGCGC CGCCACAGTA CGTTTGATCT CCAACTTGGT CCCTCCGCCG AACGTGAATG 60GAAAACTATG AACATTCTGA CAGTAGTATA CTGCCACATC TTCAGCCTGC A 111 82 basepairs nucleic acid single linear DNA (genomic) 35 ATGGTGTTGC AGACCCAGGTCTTCATTTCT CTGTTGCTCT GGATCTCTGG TGCCTACGGG 60 CAGGTTCAAC TGAAAGAGTC AG82 89 base pairs nucleic acid single linear DNA (genomic) 36 GTCCTGACTCTTTCAGTTGA ACCTGCCCGT AGGCACCAGA GATCCAGAGC AACAGAGAAA 60 TGAAGACCTGGGTCTGCAAC ACCATGTTG 89 45 base pairs nucleic acid single linear DNA(genomic) 37 CAAGGCACCA CTCTCACAGT CTCCTCAGCT AGTACGAAGG GCCCA 45 43base pairs nucleic acid single linear DNA (genomic) 38 AGCTTGGGCCCTTCGTACTA GCTGAGGAGA CTGTGAGTGG TGC 43 28 base pairs nucleic acidsingle linear DNA (genomic) 39 ATCGTGATGA CCCAGTCTCC ATCCTCCC 28 31 basepairs nucleic acid single linear DNA (genomic) 40 TCAGGGAGGA TGGAGACTGGGTCATCACGA T 31 43 base pairs nucleic acid single linear DNA (genomic)41 TCGGGGGACA GAGTTGGAAA TAAAACGTAC TGTGGCGGCG CCA 43 42 base pairsnucleic acid single linear DNA (genomic) 42 AGCTTGGCGC CGCCACAGTACGTTTTATTT CCAACTCTGT CC 42 26 base pairs nucleic acid single linear DNA(genomic) 43 CTAGCCACCA CCACCACCAC CACTAA 26 26 base pairs nucleic acidsingle linear DNA (genomic) 44 CTAGTTAGTG GTGGTGGTGG TGGTGG 26 113 aminoacids amino acid single linear protein 45 Glu Leu Val Met Thr Gln SerPro Ser Ser Leu Ser Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met SerCys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn TyrLeu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu IleTyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr GlySer Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val GlnAla Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ser Tyr ProPhe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys 113 aminoacids amino acid single linear protein 46 Glu Leu Val Met Thr Gln SerPro Ser Ser Leu Ser Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met SerCys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn TyrLeu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu IleTyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr GlySer Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val GlnAla Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr ProPhe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys 9 aminoacids amino acid single linear protein 47 Gln Asn Asp His Ser Tyr ProPhe Thr 1 5 9 amino acids amino acid single linear protein 48 Gln AsnAsp Tyr Ser Tyr Pro Phe Thr 1 5 6285 base pairs nucleic acid doublecircular DNA (genomic) 49 GACGTCGCGG CCGCTCTAGG CCTCCAAAAA AGCCTCCTCACTACTTCTGG AATAGCTCAG 60 AGGCCGAGGC GGCCTCGGCC TCTGCATAAA TAAAAAAAATTAGTCAGCCA TGCATGGGGC 120 GGAGAATGGG CGGAACTGGG CGGAGTTAGG GGCGGGATGGGCGGAGTTAG GGGCGGGACT 180 ATGGTTGCTG ACTAATTGAG ATGCATGCTT TGCATACTTCTGCCTGCTGG GGAGCCTGGG 240 GACTTTCCAC ACCTGGTTGC TGACTAATTG AGATGCATGCTTTGCATACT TCTGCCTGCT 300 GGGGAGCCTG GGGACTTTCC ACACCCTAAC TGACACACATTCCACAGAAT TAATTCCCGG 360 GGATCGATCC GTCGACGTAC GACTAGTTAT TAATAGTAATCAATTACGGG GTCATTAGTT 420 CATAGCCCAT ATATGGAGTT CCGCGTTACA TAACTTACGGTAAATGGCCC GCCTGGCTGA 480 CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGTATGTTCCCAT AGTAACGCCA 540 ATAGGGACTT TCCATTGACG TCAATGGGTG GACTATTTACGGTAAACTGC CCACTTGGCA 600 GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTGACGTCAATGA CGGTAAATGG 660 CCCGCCTGGC ATTATGCCCA GTACATGACC TTATGGGACTTTCCTACTTG GCAGTACATC 720 TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTTGGCAGTACAT CAATGGGCGT 780 GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACCCCATTGACGT CAATGGGAGT 840 TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTCGTAACAACTC CGCCCCATTG 900 ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATATAAGCAGAGC TGGGTACGTG 960 AACCGTCAGA TCGCCTGGAG ACGCCATCGA ATTCGAGGACGCCAGCAACA TGGTGTTGCA 1020 GACCCAGGTC TTCATTTCTC TGTTGCTCTG GATCTCTGGTGCCTACGGGC AGGTTACCCT 1080 GCGTGAATCC GGTCCGGCAC TAGTTAAACC GACCCAGACCCTGACGTTAA CCTGCACCGT 1140 CTCCGGTTTC TCCCTGACGA GCTATAGTGT ACACTGGGTCCGTCAGCCGC CGGGTAAAGG 1200 TCTAGAATGG CTGGGTGTAA TATGGGCTAG TGGAGGCACAGATTATAATT CGGCTCTCAT 1260 GTCCCGTCTG TCGATATCCA AAGACACCTC CCGTAACCAGGTTGTTCTGA CCATGACTAA 1320 CATGGACCCG GTTGACACCG CTACCTACTA CTGCGCTCGAGATCCCCCTT CTTCCTTACT 1380 ACGGCTTGAC TACTGGGGTC GTGGTACCCC AGTTACCGTGAGCTCAGCTA GTACCAAGGG 1440 CCCATCGGTC TTCCCCCTGG CACCCTCCTC CAAGAGCACCTCTGGGGGCA CAGCGGCCCT 1500 GGGCTGCCTG GTCAAGGACT ACTTCCCCGA ACCGGTGACGGTGTCGTGGA ACTCAGGCGC 1560 CCTGACCAGC GGCGTGCACA CCTTCCCGGC TGTCCTACAGTCCTCAGGAC TCTACTCCCT 1620 CAGCAGCGTG GTGACCGTGC CCTCCAGCAG CTTGGGCACCCAGACCTACA TCTGCAACGT 1680 GAATCACAAG CCCAGCAACA CCAAGGTGGA CAAGAGAGTTGAGCCCAAAT CTTGTGACAA 1740 AACTCACACA TGCCCACCGT GCCCAGCACC TGAACTCCTGGGGGGACCGT CAGTCTTCCT 1800 CTTCCCCCCA AAACCCAAGG ACACCCTCAT GATCTCCCGGACCCCTGAGG TCACATGCGT 1860 GGTGGTGGAC GTGAGCCACG AAGACCCTGA GGTCAAGTTCAACTGGTACG TGGACGGCGT 1920 GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAGTACAACAGCA CGTACCGTGT 1980 GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAATGGCAAGGAGT ACAAGTGCAA 2040 GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT CGAGAAAACCATCTCCAAAG CCAAAGGGCA 2100 GCCCCGAGAA CCACAGGTGT ACACCCTGCC CCCATCCCGGGAGGAGATGA CCAAGAACCA 2160 GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGCGACATCGCCG TGGAGTGGGA 2220 GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCTCCCGTGCTGG ACTCCGACGG 2280 CTCCTTCTTC CTCTATAGCA AGCTCACCGT GGACAAGAGCAGGTGGCAGC AGGGGAACGT 2340 CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCACTACACGCAGA AGAGCCTCTC 2400 CCTGTCTCCG GGTAAGTGAG TGTAGTCTAG ATCTACGTATGATCAGCCTC GACTGTGCCT 2460 TCTAGTTGCC AGCCATCTGT TGTTTGCCCC TCCCCCGTGCCTTCCTTGAC CCTGGAAGGT 2520 GCCACTCCCA CTGTCCTTTC CTAATAAAAT GAGGAAATTGCATCGCATTG TCTGAGTAGG 2580 TGTCATTCTA TTCTGGGGGG TGGGGTGGGG CAGGACAGCAAGGGGGAGGA TTGGGAAGAC 2640 AATAGCAGGC ATGCTGGGGA TGCGGTGGGC TCTATGGAACCAGCTGGGGC TCGACAGCGC 2700 TGGATCTCCC GATCCCCAGC TTTGCTTCTC AATTTCTTATTTGCATAATG AGAAAAAAAG 2760 GAAAATTAAT TTTAACACCA ATTCAGTAGT TGATTGAGCAAATGCGTTGC CAAAAAGGAT 2820 GCTTTAGAGA CAGTGTTCTC TGCACAGATA AGGACAAACATTATTCAGAG GGAGTACCCA 2880 GAGCTGAGAC TCCTAAGCCA GTGAGTGGCA CAGCATTCTAGGGAGAAATA TGCTTGTCAT 2940 CACCGAAGCC TGATTCCGTA GAGCCACACC TTGGTAAGGGCCAATCTGCT CACACAGGAT 3000 AGAGAGGGCA GGAGCCAGGG CAGAGCATAT AAGGTGAGGTAGGATCAGTT GCTCCTCACA 3060 TTTGCTTCTG ACATAGTTGT GTTGGGAGCT TGGATAGCTTGGACAGCTCA GGGCTGCGAT 3120 TTCGCGCCAA ACTTGACGGC AATCCTAGCG TGAAGGCTGGTAGGATTTTA TCCCCGCTGC 3180 CATCATGGTT CGACCATTGA ACTGCATCGT CGCCGTGTCCCAAAATATGG GGATTGGCAA 3240 GAACGGAGAC CTACCCTGGC CTCCGCTCAG GAACGAGTTCAAGTACTTCC AAAGAATGAC 3300 CACAACCTCT TCAGTGGAAG GTAAACAGAA TCTGGTGATTATGGGTAGGA AAACCTGGTT 3360 CTCCATTCCT GAGAAGAATC GACCTTTAAA GGACAGAATTAATATAGTTC TCAGTAGAGA 3420 ACTCAAAGAA CCACCACGAG GAGCTCATTT TCTTGCCAAAAGTTTGGATG ATGCCTTAAG 3480 ACTTATTGAA CAACCGGAAT TGGCAAGTAA AGTAGACATGGTTTGGATAG TCGGAGGCAG 3540 TTCTGTTTAC CAGGAAGCCA TGAATCAACC AGGCCACCTTAGACTCTTTG TGACAAGGAT 3600 CATGCAGGAA TTTGAAAGTG ACACGTTTTT CCCAGAAATTGATTTGGGGA AATATAAACT 3660 TCTCCCAGAA TACCCAGGCG TCCTCTCTGA GGTCCAGGAGGAAAAAGGCA TCAAGTATAA 3720 GTTTGAAGTC TACGAGAAGA AAGACTAACA GGAAGATGCTTTCAAGTTCT CTGCTCCCCT 3780 CCTAAAGCTA TGCATTTTTA TAAGACCATG GGACTTTTGCTGGCTTTAGA TCAGCCTCGA 3840 CTGTGCCTTC TAGTTGCCAG CCATCTGTTG TTTGCCCCTCCCCCGTGCCT TCCTTGACCC 3900 TGGAAGGTGC CACTCCCACT GTCCTTTCCT AATAAAATGAGGAAATTGCA TCGCATTGTC 3960 TGAGTAGGTG TCATTCTATT CTGGGGGGTG GGGTGGGGCAGGACAGCAAG GGGGAGGATT 4020 GGGAAGACAA TAGCAGGCAT GCTGGGGATG CGGTGGGCTCTATGGAACCA GCTGGGGCTC 4080 GATCGAGTGT ATGACTGCGG CCGCGATCCC GTCGAGAGCTTGGCGTAATC ATGGTCATAG 4140 CTGTTTCCTG TGTGAAATTG TTATCCGCTC ACAATTCCACACAACATACG AGCCGGAAGC 4200 ATAAAGTGTA AAGCCTGGGG TGCCTAATGA GTGAGCTAACTCACATTAAT TGCGTTGCGC 4260 TCACTGCCCG CTTTCCAGTC GGGAAACCTG TCGTGCCAGCTGCATTAATG AATCGGCCAA 4320 CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGCTCTTCCGCTTCCTCGCT CACTGACTCG 4380 CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGCTCACTCAAAGGC GGTAATACGG 4440 TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACATGTGAGCAAAAGG CCAGCAAAAG 4500 GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTTCCATAGGCTCCG CCCCCCTGAC 4560 GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAAACCCGACAGG ACTATAAAGA 4620 TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTCTCCTGTTCCGAC CCTGCCGCTT 4680 ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGGCGCTTTCTCA ATGCTCACGC 4740 TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAAGCTGGGCTGTGT GCACGAACCC 4800 CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATCGTCTTGAGTC CAACCCGGTA 4860 AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAACAGGATTAGCAG AGCGAGGTAT 4920 GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACTACGGCTACAC TAGAAGGACA 4980 GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTTCGGAAAAAGAGT TGGTAGCTCT 5040 TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTTTTGTTTGCAA GCAGCAGATT 5100 ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGATCTTTTCTACGGG GTCTGACGCT 5160 CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGAGATTATCAAA AAGGATCTTC 5220 ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAATCTAAAGTAT ATATGAGTAA 5280 ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGCACCTATCTCAGC GATCTGTCTA 5340 TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGATAACTACGAT ACGGGAGGGC 5400 TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGACCCACGCTCACC GGCTCCAGAT 5460 TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCAGAAGTGGTCC TGCAACTTTA 5520 TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTAGAGTAAGTAG TTCGCCAGTT 5580 AATAGTTTGC GCAACGTTGT TGCCATTGCT ACAGGCATCGTGGTGTCACG CTCGTCGTTT 5640 GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGCGAGTTACATG ATCCCCCATG 5700 TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCGTTGTCAGAAG TAAGTTGGCC 5760 GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATTCTCTTACTGT CATGCCATCC 5820 GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGTCATTCTGAGA ATAGTGTATG 5880 CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGATAATACCGCGCC ACATAGCAGA 5940 ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGCGAAAACTCTC AAGGATCTTA 6000 CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCACCCAACTGATC TTCAGCATCT 6060 TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAAGGCAAAATGC CGCAAAAAAG 6120 GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCTTCCTTTTTCA ATATTATTGA 6180 AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATATTTGAATGTAT TTAGAAAAAT 6240 AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGCCACCT 6285 5703 base pairs nucleic acid double circular DNA (genomic) 50GACGTCGCGG CCGCTCTAGG CCTCCAAAAA AGCCTCCTCA CTACTTCTGG AATAGCTCAG 60AGGCCGAGGC GGCCTCGGCC TCTGCATAAA TAAAAAAAAT TAGTCAGCCA TGCATGGGGC 120GGAGAATGGG CGGAACTGGG CGGAGTTAGG GGCGGGATGG GCGGAGTTAG GGGCGGGACT 180ATGGTTGCTG ACTAATTGAG ATGCATGCTT TGCATACTTC TGCCTGCTGG GGAGCCTGGG 240GACTTTCCAC ACCTGGTTGC TGACTAATTG AGATGCATGC TTTGCATACT TCTGCCTGCT 300GGGGAGCCTG GGGACTTTCC ACACCCTAAC TGACACACAT TCCACAGAAT TAATTCCCGG 360GGATCGATCC GTCGACGTAC GACTAGTTAT TAATAGTAAT CAATTACGGG GTCATTAGTT 420CATAGCCCAT ATATGGAGTT CCGCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA 480CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT AGTAACGCCA 540ATAGGGACTT TCCATTGACG TCAATGGGTG GACTATTTAC GGTAAACTGC CCACTTGGCA 600GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG ACGTCAATGA CGGTAAATGG 660CCCGCCTGGC ATTATGCCCA GTACATGACC TTATGGGACT TTCCTACTTG GCAGTACATC 720TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT 780GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT 840TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG 900ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA TAAGCAGAGC TGGGTACGTG 960AACCGTCAGA TCGCCTGGAG ACGCCATCGA ATTCATTGAT AGGATCCAGC AAGATGGTGT 1020TGCAGACCCA GGTCTTCATT TCTCTGTTGC TCTGGATCTC TGGTGCCTAC GGGGATATCG 1080TGATGACCCA GTCTCCAGAC TCGCTAGCTG TGTCTCTGGG CGAGAGGGCC ACCATCAACT 1140GCAAGAGCTC TCAGAGTCTG TTAAACAGTG GAAATCAAAA GAACTACTTG GCCTGGTATC 1200AGCAGAAACC CGGGCAGCCT CCTAAGTTGC TCATTTACGG GGCGTCGACT AGGGAATCTG 1260GGGTACCTGA CCGATTCAGT GGCAGCGGGT CTGGGACAGA TTTCACTCTC ACCATCAGCA 1320GCCTGCAGGC TGAAGATGTG GCAGTATACT ACTGTCAGAA TGTTCATAGT TTTCCATTCA 1380CGTTCGGCGG AGGGACCAAG TTGGAGATCA AACGTACTGT GGCGGCGCCA TCTGTCTTCA 1440TCTTCCCGCC ATCTGATGAG CAGTTGAAAT CTGGAACTGC CTCTGTTGTG TGCCTGCTGA 1500ATAACTTCTA TCCCAGAGAG GCCAAAGTAC AGTGGAAGGT GGATAACGCC CTCCAATCGG 1560GTAACTCCCA GGAGAGTGTC ACAGAGCAGG ACAGCAAGGA CAGCACCTAC AGCCTCAGCA 1620GCACCCTGAC GCTGAGCAAA GCAGACTACG AGAAACACAA AGTCTACGCC TGCGAAGTCA 1680CCCATCAGGG CCTGAGCTCG CCCGTCACAA AGAGCTTCAA CAGGGGAGAG TGTTAATTCT 1740AGATCCGTTA TCTACGTATG ATCAGCCTCG ACTGTGCCTT CTAGTTGCCA GCCATCTGTT 1800GTTTGCCCCT CCCCCGTGCC TTCCTTGACC CTGGAAGGTG CCACTCCCAC TGTCCTTTCC 1860TAATAAAATG AGGAAATTGC ATCGCATTGT CTGAGTAGGT GTCATTCTAT TCTGGGGGGT 1920GGGGTGGGGC AGGACAGCAA GGGGGAGGAT TGGGAAGACA ATAGCAGGCA TGCTGGGGAT 1980GCGGTGGGCT CTATGGAACC AGCTGGGGCT CGACAGCTCG AGCTAGCTTT GCTTCTCAAT 2040TTCTTATTTG CATAATGAGA AAAAAAGGAA AATTAATTTT AACACCAATT CAGTAGTTGA 2100TTGAGCAAAT GCGTTGCCAA AAAGGATGCT TTAGAGACAG TGTTCTCTGC ACAGATAAGG 2160ACAAACATTA TTCAGAGGGA GTACCCAGAG CTGAGACTCC TAAGCCAGTG AGTGGCACAG 2220CATTCTAGGG AGAAATATGC TTGTCATCAC CGAAGCCTGA TTCCGTAGAG CCACACCTTG 2280GTAAGGGCCA ATCTGCTCAC ACAGGATAGA GAGGGCAGGA GCCAGGGCAG AGCATATAAG 2340GTGAGGTAGG ATCAGTTGCT CCTCACATTT GCTTCTGACA TAGTTGTGTT GGGAGCTTGG 2400ATCGATCCAC CATGGTTGAA CAAGATGGAT TGCACGCAGG TTCTCCGGCC GCTTGGGTGG 2460AGAGGCTATT CGGCTATGAC TGGGCACAAC AGACAATCGG CTGCTCTGAT GCCGCCGTGT 2520TCCGGCTGTC AGCGCAGGGG CGCCCGGTTC TTTTTGTCAA GACCGACCTG TCCGGTGCCC 2580TGAATGAACT GCAGGACGAG GCAGCGCGGC TATCGTGGCT GGCCACGACG GGCGTTCCTT 2640GCGCAGCTGT GCTCGACGTT GTCACTGAAG CGGGAAGGGA CTGGCTGCTA TTGGGCGAAG 2700TGCCGGGGCA GGATCTCCTG TCATCTCACC TTGCTCCTGC CGAGAAAGTA TCCATCATGG 2760CTGATGCAAT GCGGCGGCTG CATACGCTTG ATCCGGCTAC CTGCCCATTC GACCACCAAG 2820CGAAACATCG CATCGAGCGA GCACGTACTC GGATGGAAGC CGGTCTTGTC GATCAGGATG 2880ATCTGGACGA AGAGCATCAG GGGCTCGCGC CAGCCGAACT GTTCGCCAGG CTCAAGGCGC 2940GCATGCCCGA CGGCGAGGAT CTCGTCGTGA CCCATGGCGA TGCCTGCTTG CCGAATATCA 3000TGGTGGAAAA TGGCCGCTTT TCTGGATTCA TCGACTGTGG CCGGCTGGGT GTGGCGGACC 3060GCTATCAGGA CATAGCGTTG GCTACCCGTG ATATTGCTGA AGAGCTTGGC GGCGAATGGG 3120CTGACCGCTT CCTCGTGCTT TACGGTATCG CCGCTCCCGA TTCGCAGCGC ATCGCCTTCT 3180ATCGCCTTCT TGACGAGTTC TTCTGAGCGG GACTCTGGGG TTCGAAATGA CCGACCAAGC 3240GACGCCCAAC CTGCCATCAC GAGATTTCGA TTCCACCGCC GCCTTCTATG AAAGGTTGGG 3300CTTCGGAATC GTTTTCCGGG ACGCCGGCTG GATGATCCTC CAGCGCGGGG ATCTCATGCT 3360GGAGTTCTTC GCCCACCCCA ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA 3420TAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC 3480CAAACTCATC AATGTATCTT ATCATGTCTG GATCGCGGCC GCGATCCCGT CGAGAGCTTG 3540GCGTAATCAT GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCAC AATTCCACAC 3600AACATACGAG CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT GAGCTAACTC 3660ACATTAATTG CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC GTGCCAGCTG 3720CATTAATGAA TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCG CTCTTCCGCT 3780TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC 3840TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA 3900GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT 3960AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC 4020CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT 4080GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG 4140CTTTCTCAAT GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG 4200GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT 4260CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG 4320ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC 4380GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA 4440AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT 4500GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT 4560TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA 4620TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC 4680TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT 4740ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA 4800ACTACGATAC GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA 4860CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA 4920AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA 4980GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTAC AGGCATCGTG 5040GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA 5100GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT 5160GTCAGAAGTA AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT 5220CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA 5280TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAT ACGGGATAAT 5340ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA 5400AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC 5460AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG 5520CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC 5580CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT 5640GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA 5700CCT 5703 81 base pairs nucleic acid single linear DNA (genomic) 51ATCCAAAGAC AACTCCCGTA ACCAGGTTGT TCTGACCATG ACTAACATGG ACCCGGTTGA 60CACCGCTACC TACTACTGCG C 81 85 base pairs nucleic acid single linear DNA(genomic) 52 TCGAGCGCAG TAGTAGGTAG CGGTGTCAAC CGGGTCCATG TTAGTCATGGTCAGAACAAC 60 CTGGTTACGG GAGTTGTCTT TGGAT 85 73 base pairs nucleic acidsingle linear DNA (genomic) 53 AACCTGCACC GTCTCCGGTT TCTCCCTGACGAGCTATAGT GTACACTGGA TCCGTCAGCC 60 GCCGGGTAAA GGT 73 77 base pairsnucleic acid single linear DNA (genomic) 54 CTAGACCTTT ACCCGGCGGCTGACGGATCC AGTGTACACT ATAGCTCGTC AGGGAGAAAC 60 CGGAGACGGT GCAGGTT 77 46base pairs nucleic acid single linear DNA (genomic) 55 CTAGCTGTGTCAGCTGGCGA GAGGGCCACC ATCAACTGCA AGAGCT 46 38 base pairs nucleic acidsingle linear DNA (genomic) 56 CTTGCAGTTG ATGGTGGCCC TCTCGCCAGC TGACACAG38 140 base pairs nucleic acid single linear DNA (genomic) 57 TTCGAGGACGCCAGCAACAT GGTGTTGCAG ACCCAGGTCT TCATTTCTCT GTTGCTCTGG 60 ATCTCTGGTGCCTACGGGCA GGTCCAACTG CAGGAGAGCG GTCCAGGTCT TGTGAGACCT 120 AGCCAGACCCTGAGCCTGAC 140 138 base pairs nucleic acid single linear DNA (genomic)58 GTGCCTCCAC TAGCCCATAT TACTCCAAGC CACTCTAGAC CTCGTCCAGG TGGCTGTCTC 60ACCCAGTGTA CACTATAGCT GGTGAGGGAG AAGCCCGAGA CGGTGCAGGT CAGGCTCAGG 120GTCTGGCTAG GTCTCACA 138 143 base pairs nucleic acid single linear DNA(genomic) 59 GGCTTGGAGT AATATGGGCT AGTGGAGGCA CAGATTATAA TTCGGCTCTCATGTCCAGAC 60 TGAGTATACT GAAAGACAAC AGCAAGAACC AGGTCAGCCT GAGACTCAGCAGCGTGACAG 120 CCGCCGACAC CGCGGTCTAT TTC 143 136 base pairs nucleic acidsingle linear DNA (genomic) 60 CCAGTGCCAA GCTTGGGCCC TTGGTGGAGGCGCTCGAGAC GGTGACCGTG GTACCTTGTC 60 CCCAGTAGTC AAGCCGTAGT AAGGAAGAAGGGGGATCTCG AGCACAGAAA TAGACCGCGG 120 TGTCGGCGGC TGTCAC 136 357 basepairs nucleic acid double linear DNA (genomic) 61 CAGGTCCAAC TGCAGGAGAGCGGTCCAGGT CTTGTGAGAC CTAGCCAGAC CCTGAGCCTG 60 ACCTGCACCG TCTCGGGCTTCTCCCTCACC AGCTATAGTG TACACTGGGT GAGACAGCCA 120 CCTGGACGAG GTCTAGAGTGGCTTGGAGTA ATATGGGCTA GTGGAGGCAC AGATTATAAT 180 TCGGCTCTCA TGTCCAGACTGAGTATACTG AAAGACAACA GCAAGAACCA GGTCAGCCTG 240 AGACTCAGCA GCGTGACAGCCGCCGACACC GCGGTCTATT ACTGTGCTCG GGATCCCCCT 300 TCTTCCTTAC TACGGCTTGACTACTGGGGA CAAGGTACCA CGGTCACCGT CTCGAGC 357 119 amino acids amino acidsingle linear protein 62 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu ValArg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly PheSer Leu Thr Ser Tyr 20 25 30 Ser Val His Trp Val Arg Gln Pro Pro Gly ArgGly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ala Ser Gly Gly Thr Asp TyrAsn Ser Ala Leu Met 50 55 60 Ser Arg Leu Ser Ile Leu Lys Asp Asn Ser LysAsn Gln Val Ser Leu 65 70 75 80 Arg Leu Ser Ser Val Thr Ala Ala Asp ThrAla Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Pro Pro Ser Ser Leu Leu Arg LeuAsp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 28base pairs nucleic acid single linear DNA (genomic) 63 AGGACGCCAGCAACATGGTG TTGCAGAC 28 36 base pairs nucleic acid single linear DNA(genomic) 64 TGCCAAGCTT GGGCCCTTGG TGGAGGCGCT CGAGAC 36 121 base pairsnucleic acid single linear DNA (genomic) 65 GACCATGATT ACGAATTCGTAGTCGGATAT CGTGATGACC CAGAGCCCAA GCAGCCTGAG 60 CGCTAGCGTG GGTGACAGAGTGACCATCAC CTGTAAGAGC TCTCAGAGTC TGTTAAACAG 120 T 121 116 base pairsnucleic acid single linear DNA (genomic) 66 AGATTCCCTA GTCGATGCCCCGTAGATCAG CAGCTTTGGA GCCTTACCGG GTTTCTGCTG 60 ATACCAGGCC AAGTAGTTCTTTTGATTTCC ACTGTTTAAC AGACTCTGAG AGCTCT 116 116 base pairs nucleic acidsingle linear DNA (genomic) 67 TCTACGGGGC ATCGACTAGG GAATCTGGGGTACCAGATAG ATTCAGCGGT AGCGGTAGCG 60 GAACCGACTT CACCTTCACC ATCAGCAGCCTGCAGCCAGA GGACATCGCC ACCTAC 116 117 base pairs nucleic acid singlelinear DNA (genomic) 68 TCGATGCCAA GCTTGGCGCC GCCACAGTAC GTTTGATCTCCACCTTGGTC CCTTGTCCGA 60 ACGTGAATGG AAAACTATGA ACATTCTGGC AGTAGTAGGTGGCGATGTCC TCTGGCT 117 339 base pairs nucleic acid double linear DNA(genomic) 69 GATATCGTGA TGACCCAGAG CCCAAGCAGC CTGAGCGCTA GCGTGGGTGACAGAGTGACC 60 ATCACCTGTA AGAGCTCTCA GAGTCTGTTA AACAGTGGAA ATCAAAAGAACTACTTGGCC 120 TGGTATCAGC AGAAACCCGG TAAGGCTCCA AAGCTGCTGA TCTACGGGGCATCGACTAGG 180 GAATCTGGGG TACCAGATAG ATTCAGCGGT AGCGGTAGCG GAACCGACTTCACCTTCACC 240 ATCAGCAGCC TGCAGCCAGA GGACATCGCC ACCTACTACT GCCAGAATGTTCATAGTTTT 300 CCATTCACGT TCGGACAAGG GACCAAGGTG GAGATCAAA 339 113 aminoacids amino acid single linear protein 70 Asp Ile Val Met Thr Gln SerPro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile ThrCys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn TyrLeu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu IleTyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser GlySer Gly Ser Gly Thr Asp Phe Thr Phe Thr 65 70 75 80 Ile Ser Ser Leu GlnPro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn 85 90 95 Val His Ser Phe ProPhe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys 24 basepairs nucleic acid single linear DNA (genomic) 71 GATTACGAAT TCGTAGTCGGATAT 24 24 base pairs nucleic acid single linear DNA (genomic) 72TGCCAAGCTT GGCGCCGCCA CAGT 24 39 base pairs nucleic acid single linearDNA (genomic) 73 CTAGTGCGGG TGACCGAGTG ACCATCACCT GTAAGAGCT 39 31 basepairs nucleic acid single linear DNA (genomic) 74 CTTACAGGTG ATGGTCACTCGGTCACCCGC A 31 66 base pairs nucleic acid single linear DNA (genomic)75 GGTCTATTAC TGTGCTCGGG ATCCCCCTTC TTCCTTACTA CGGCTTGACT ACTGGGGACA 60AGGTAC 66 64 base pairs nucleic acid single linear DNA (genomic) 76CTTGTCCCCA GTAGTCAAGC CGTAGTAAGG AAGAAGGGGG ATCCCGAGCA CAGTAATAGA 60CCGC 64

What is claimed is:
 1. A rodent neutralizing monoclonal antibodyspecific for human interleukin-5 and having a binding affinitycharacterized by a dissociation constant equal to or less than about3.5×10⁻¹¹ M.
 2. The monoclonal antibody according to claim 1 which is amurine monoclonal antibody.
 3. The monoclonal antibody according toclaim 1 which is a rat monoclonal antibody.
 4. The monoclonal antibodyaccording to claim 2 which comprises the light chain amino acid sequenceof SEQ ID NO: 16, and the heavy chain amino acid sequence of SEQ ID NO:15.
 5. The monoclonal antibody according to claim 1 having theidentifying characteristics of 2B6, 2E3, 2F2 or 4A6.
 6. A hybridomahaving the identifying characteristics of cell line SK119-2B6.206.75(1),SK119-2E3.39.40.2, SK119-2F2.37.80.12 or 4A6(1)G1F7.
 7. A neutralizingFab fragment or F(ab′)₂ fragment thereof, produced by deleting the Fcregion of the monoclonal antibody of claim
 1. 8. A neutralizing Fabfragment or F(ab′)₂ fragment thereof, produced by chain shufflingwhereby the Fd heavy chain of the monoclonal antibody of claim 1 isexpressed in a murine light chain filamentous phage Fab display library.9. A neutralizing Fab fragment or F(ab′)₂ fragment thereof, produced bychain shuffling whereby the light chain of the monoclonal antibody ofclaim 1 is expressed in a murine heavy chain filamentous phage Fabdisplay library.
 10. An altered antibody comprising a heavy chain and alight chain, wherein the framework regions of said heavy and lightchains are derived from at least one selected antibody and the aminoacid sequences of the complementarity determining regions of each saidchain are derived from the monoclonal antibody of claim
 1. 11. Thealtered antibody according to claim 10 wherein said amino acid sequencesof the complementarity determining regions of the heavy chain are: (a)SEQ ID NO: 7, 8, 9; or (b) SEQ ID NO: 7, 8
 14. 12. The altered antibodyaccording to claim 10 wherein said amino acid sequences of thecomplementarity determining regions of the light chain are: (a) SEQ IDNO: 10, 11, 12; or (b) SEQ ID NO: 10, 11,
 13. 13. The altered antibodyaccording to claim 10 wherein said framework regions of said heavy chaincomprise: amino acids 1-30, 36-49, 66-97 and 109-119 of SEQ ID NO: 19.14. The altered antibody according to claim 10 wherein said frameworkregions of said light chain comprise: amino acids 1-23, 41-55, 63-94 and104-113 of SEQ ID NO:
 21. 15. An immunoglobulin heavy chaincomplementarity determining region (CDR), the amino acid sequence ofwhich is selected from the group consisting of: (a) SEQ ID NO: 7, (b)SEQ ID NO: 8, (c) SEQ ID NO: 9, and (d) SEQ ID NO:
 14. 16. Animmunoglobulin light chain complementarity determining region (CDR), theamino acid sequence of which is selected from the group consisting of:(a) SEQ ID NO: 10, (b) SEQ ID NO: 11, (c) SEQ ID NO: 12, (d) SEQ ID NO:13, (e) SEQ ID NO: 47, and (f) SEQ ID NO:
 48. 17. A nucleic acidmolecule encoding the immunoglobulin complementarity determining region(CDR) of claim
 15. 18. A nucleic acid molecule encoding theimmunoglobulin complementarity determining region (CDR) of claim
 16. 19.A chimeric antibody comprising a heavy chain and a light chain, saidantibody characterized by a dissociation constant equal or less thanabout 3.5×10⁻¹¹ M for human interleukin-5, wherein the constant regionsof said heavy and light chains are derived from at least one selectedantibody and the amino acid sequences of the variable regions of eachsaid chain are derived from the monoclonal antibody of claim
 1. 20. Theantibody according to claim 19 wherein the constant regions are selectedfrom human immunoglobulins.
 21. A pharmaceutical composition comprisingthe altered antibody of claim 10 and a pharmaceutically acceptablecarrier.
 22. A method of treating conditions associated with excesseosinophil production in a human comprising the step of administering tosaid human in need thereof an effective amount of the altered antibodyof claim
 10. 23. The method of claim 22 wherein said conditionassociated with excess eosinophil production is asthma.
 24. The methodof claim 22 wherein said condition associated with excess eosinophilproduction is allergic rhinitis.
 25. The method of claim 22 wherein saidcondition associated with excess eosinophil production is atopicdermatitis.
 26. An isolated nucleic acid sequence which is selected fromthe group consisting of: (a) a nucleic acid sequence encoding thealtered antibody of claim 10; (b) a nucleic acid sequence complementaryto (a); and (c) a fragment or analog of (a) or (b) which encodes aprotein characterized by having a specificity for human interleukin-5;wherein said sequence optionally contains a restriction site.
 27. Theisolated nucleic acid sequence according to claim 26, wherein thesequence encoding the humanized heavy chain variable region whichcomprises the nucleic acid sequence of SEQ ID NO:
 18. 28. The isolatednucleic acid sequence according to claim 26, wherein the sequenceencoding the humanized heavy chain variable region which comprises thenucleic acid sequence of SEQ ID NO:
 61. 29. The isolated nucleic acidsequence according to claim 26, wherein the sequence encoding thehumanized light chain variable region which comprises the nucleic acidsequence of SEQ ID NO:
 20. 30. The isolated nucleic acid sequenceaccording to claim 26, wherein the sequence encoding the humanized lightchain variable region which comprises the nucleic acid sequence of SEQID NO:
 69. 31. A recombinant plasmid comprising the nucleic acidsequence of claim
 26. 32. A host cell transfected with the recombinantplasmid of claim
 31. 33. A process for producing an altered antibodyspecific for human interleukin-5 comprising culturing a cell linetransfected with the recombinant plasmid of claim 31 under the controlof selected regulatory sequences capable of directing the expressionthereof in said cells.
 34. A method to assess the presence or absence ofhuman IL-5 in a human which comprises obtaining a sample of biologicalfluid from a patient and allowing the monoclonal antibody of claim 1 tocome in contact with such sample under conditions such that anIL-5/monoclonal antibody complex can form and detecting the presence orabsence of said IL-5/monoclonal antibody complex.
 35. A method foraiding in the diagnosis of allergies and other conditions associatedwith excess eosinophil production comprising the steps of determiningthe amount of human IL-5 in a sample of a patient according to themethod of claim 34 and comparing that to the mean amount of human IL-5in the normal population, whereby the presence of significantly elevatedamount of human IL-5 in the patient is an indication of allergies andother conditions associated with excess eosinophil production.
 36. Ahybridoma having the identifying characteristics of cell lineSK119-24G9.8.20.5 or 5D3(1)F5D6.
 37. A monoclonal antibody produced bythe hybridoma of claim 36.